Polymeric supports

ABSTRACT

Azlactone-functional polymer supports are useful reactive supports for the attachment of functional materials to provide novel adduct beads. The adduct beads are useful as complexing agents, catalysts, polymeric reagents, chromatographic supports, and as enzyme- or other biologically active supports. Novel carboxylate-functional polymer beads, are intermediates in the preparation of the azlactone-functional beads. 
     Azlactone-functional supports have units of the formula: ##STR1## wherein R 1  is H or CH 3 , 
     R 2  and R 3  independently can be an alkyl group having 1 to 14 carbon atoms, a cycloalkyl group having 3 to 14 carbon atoms, an aryl group having 5 to 12 ring atoms, an arenyl group having 6 to 26 carbon and 0 to 3 S, N, and nonperoxidic O heteroatoms, or R 2  and R 3  taken together with the carbon to which they are joined can form a carbocyclic ring containing 4 to 12 ring atoms, and 
     n is an integer 0 or 1, 
     the azlactone functional supports having 0.1 to 99 molar parts of crosslinking monomer incorporated therein.

This is a continuation of application Ser. No. 07/335,835 filed Apr. 10,1989, now U.S. Pat. No. 5,292,840, which is a continuation-in-part ofapplication Ser. No. 07/158,258 filed Feb. 19, 1988, now U.S. Pat. No.4,871,824, which is a continuation-in-part of application Ser. No.07/025,605, filed Mar. 13, 1987, now U.S. Pat. No. 4,737,560.

FIELD OF THE INVENTION

This invention relates to azlactone-functional supports, includingpolymer beads, membranes, films, and coatings. The azlactone-functionalsupports are useful for attachment of functional materials to providenovel adduct supports. The adduct supports are useful as complexingagents, catalysts, and polymeric reagents, as enzyme or otherprotein-bearing supports, and as chromatographic supports. In additionalaspects, methods of preparation of the supports are disclosed.

BACKGROUND OF THE INVENTION

The attachment of useful materials such as catalysts, reagents,chelating or complexing agents, and proteins to insoluble supports iswell-known. With the attending advantages of ease of removal andrecovery from the system, e.g., by simple filtration, regeneration (ifnecessary), and recycling coupled with the increased utilization ofcontinuous flow systems in both general chemical processing anddiagnostic monitoring procedures, supported materials are ubiquitous intoday's technology. One indication of this is the listing of "Polymer-Supported Reagents" as a separate heading in the General Subjects Indexof Chemical Abstracts beginning in 1982.

Concerning the nature of the insoluble support material, both inorganicpolymers (notably silica gel and alumina) and organic polymers have beenutilized. Factors, however, such as increased capacity because of betterporosity (especially with the so-called "gel-type" polymers which swellsomewhat and allow relatively free access by solvent and solute to thebound functionality within the support) and better control of the polarnature of the support (by selection of appropriate comonomers), whichhas been shown to directly affect reaction rate, have led to a generalpreference for the organic polymer supports. Polystyrene has been thesolid support material most extensively utilized.

The attaching functionality for polystyrene supports most often utilizedhas been the chloromethylphenyl group. These reactive, solid supportsare the so-called "Merrifield resins", so named for R. B. Merrifield (J.Am. Chem. Soc., 85, 2149 (1963)) who received the Nobel Prize inChemistry in 1984 for these and other achievements. Merrifield resinsare extremely useful for conducting solid phase peptide syntheses, buttheir broad utilization as reactive, solid supports is limited becauseof the relative nonpolarity of the hydrophobic polystyrene backbone, anoftentimes unpredictable attaching reaction which involves nucleophilicdisplacement of chloride ion, and a relatively low capacity of reactablechloromethylphenyl groups per gram of polymer. The chloromethylphenyland other reactive functionalities are discussed by N. K. Mathur, C. K.Narang, and R. E. Williams, "Polymers as Aids in Organic Chemistry",Chapter 2, Academic Press: New York (1980).

The present state of reactive, insoluble supports may be summarized bythe statement that no one support is broadly suitable for the manyapplications of solid-supported materials. The spectrum of propertiesrequired varies tremendously depending on the end-use, which includessuch diverse applications as mediating organic synthetictransformations, removing precious metals from sea water or heavy metalcontaminants from industrial effluants, utilizing supported metals ascatalysts for conducting organic reactions and polymerizations,resolving optical isomers, separating biomacromolecules, and attachingbiomacromolecules.

Azlactones have not been previously utilized as attaching groups oninsoluble supports. Azlactones have, however, been proposed to be usefulin two instances.

U.S. Pat. No. 4,070,348 teaches the preparation of water-swellable,crosslinked bead copolymers having 0.2 to 5 mol percent crosslinkingmonomer and at least 10 mole percent of a water soluble comonomerincorporated therein. The copolymers are reactive with proteinsprimarily by the inclusion of oxirane groups which are the only reactivegroups claimed. Several "activated carboxyl groups" (col. 4; line 42),however, are listed including a 2-alkenyl azlactone,2-isopropenyl-4,4-dimethyl-oxazolone-5 (col. 5; lines 2-3), and reactionof this compound with a primary amino group of a protein is depictedschematically (col. 5; lines 6-14). No additional information orenabling disclosure is given about incorporation of the azlactone into ahydrophilic, crosslinked bead copolymer or reaction of anazlactone-functional insoluble support with a protein or any otherfunctional material. The crosslinked, bead copolymers of U.S. Pat. No.4,070,348 are all prepared purposely in essentially an anhydrouscondition, i.e. with care being taken to exclude water.

L. D. Taylor, et al., Makromol. Chem., Rapid Commun., 3, 779 (1982) haveproposed azlactones to be useful as reactive groups on polymericsupports. Only the bulk homopolymerization of2-vinyl-4,4-dimethylazlactone to form a polymeric "plug" is described.No mention of crosslinking and generation of polymeric beads is given.Furthermore, described at some length is the susceptibility of thepoly(azlactone) to hydrolysis, i.e., ring-opening reaction with water[equation (1)]. Hydrolysis is regarded as being very facile, occurringeven with traces of moisture often present in organic solvents for thehomopolymer, as follows: ##STR2## Based on this account of thepropensity toward hydrolysis, it is entirely unexpected that anazlactone-functional support could be selectively reacted with afunctional material in aqueous media.

SUMMARY OF THE INVENTION

Briefly, the present invention provides hydrophilic azlactone-functionalsupports, including polymer beads, membranes, films, and coatings,having in the range of 0 to 99 molar parts of crosslinking monomerincorporated therein.

In another aspect, the present invention provides novel adduct supportswhich are produced by a ring opening reaction between theazlactone-functional supports of the invention and functional materials.The adduct supports are useful as complexing agents, catalysts,reagents, adsorbants, chromatographic supports, and as biologicallyactive supports.

The present invention provides four methods for the preparation ofsupports of the invention. Several methods are available for preparingazlactone-functional supports. One method is to apply alkenyl azlactonemonomer to the support (optionally along with other co-monomers) andpolymerize the monomer(s) in place, e.g., by photopolymerization(utilizing an appropriate photoinitiator).

In Process I carboxylate-functional polymer supports are prepared asintermediates to azlactone-functional polymer supports by the reversephase suspension polymerization product of:

(i) optionally, at least one free radically addition polymerizable,water soluble monomer,

(ii) at least one water-soluble salt of an N-(meth)acryloylamino acid,and

(iii) at least one crosslinking monomer.

Reaction of a cyclization agent and the carboxylate-functional polymersupports just described provides azlactone-functional polymer supports.

In Process II of the invention the azlactone-functional supports can beprepared by the reverse phase suspension polymerization product of

(i) optionally, at least one free radically addition polymerizable,water soluble monomer,

(ii) at least one alkenyl azlactone, and

(iii) at least one crosslinking monomer.

In Process III of the invention the azlactone-functional supports can beprepared by the dispersion polymerization reaction product of

(i) optionally, at least one free radically addition polymerizablemonomer,

(ii) at least one alkenyl azlactone, and

(iii) optionally, at least one crosslinking monomer.

In Process IV of the invention azlactone-functional supports can beprovided by coating an azlactone-functional polymer onto a solidsupport.

Reaction of the azlactone-functional supports of the invention withfunctional materials capable of reacting with the azlactone ring (i.e,by a ring-opening reaction) provides the adduct supports of theinvention. We have discovered that this adduct-forming reaction occursto a high degree with a dissolved nucleophile in water solution,especially when the nucleophile is primary amine-functional. Thisselectivity of reaction is even more surprising when one considers thatthe concentration of the amine nucleophile on a protein functionalmaterial, for example, is most often substantially lower than that ofthe water solvent. Before the present invention, it was thought thatazlactone groups would predominantly react with water, i.e., hydrolyze,rather than react with a dissolved nucleophile.

The hydrophilic or hydrophobic nature of a support is extremelyimportant in determining its utility. An obvious advantage of ahydrophilic support is that many of the operations of supportedmaterials are conducted in aqueous media. Water is virtually theexclusive solvent for conducting precious or noxious metal ion removal,in diagnostic monitoring of components of biofluids and biosystems, aswell as in a number of chemical reactions, and it is oftentimesadvantageous to utilize a polymer support which will swell in water. Thewater solvent can facilitate the additional encounter and interaction ofa solute and reactive groups within the hydrophilic support as well asat the support-water interface.

Hydrophilic polymer-supported materials find use and are beneficial innon-aqueous systems as well. Functional groups which imparthydrophilicity are highly polar in nature, and supported materialfunctions which are sensitive to solvent effects will be tremendouslyaffected, especially in terms of rate, by the polarity of the polymerbackbone. Importance of the polymer backbone in determining the localenvironment for a supported material has been noted by H. Morawetz, J.Macromol. Sci.--Chem., A-13, 311 (1979).

As has been noted above, U.S. Pat. No. 4,070,348 discloseswater-swellable, crosslinked bead copolymers having 0.2 to 5 mol percentcrosslinking monomer and at least 10 mole percent of a water solublecomonomer incorporated therein. The patentee desires beads having a highdegree of swelling in water, i.e., 5-100 times as is disclosed in col.6, lines 66-67. This high degree of swelling is deemed important toachieve high binding capacity with proteins. In col. 9, lines 30-32, ofU.S. Pat. No. 4,070,348, it is stated that "The greatest part of thebiologically active substances are found in the wide mesh `hollowspaces` within the swollen particles."

However, many applications, particularly chromatographic applications,cannot conveniently utilize support materials which exhibit a highdegree of swelling in aqueous media.

Surprisingly, we have now found that azlactone beads having remarkablyhigh binding capacity with functional materials can be achieved withhighly crosslinked beads which swell very modestly, e.g., threefold orless, in water. When, desired, a high degree of crosslinking is achievedby incorporating greater than 5 and up to 99 molar parts (mol percent)crosslinking monomer, preferably 7 to 99 molar parts, more preferably 10to 99 molar parts, and most preferably 30 to 99 molar parts of at leastone crosslinking monomer into the azlactone-functional polymer beads.

In this application:

"azlactone-functional support" means an article comprising anazlactone-functional polymer or an azlactone-functional polymer coatedon at least one surface of a substrate;

"acryloyl" means not only 1-oxo-2-propenyl but also1-oxo-2-methyl-2-propenyl resulting from methacryloylation reactions;

"alkyl" means the monovalent residue remaining after removal of ahydrogen atom from a saturated linear or branched chain hydrocarbonhaving 1 to 14 carbon atoms;

"aryl" means the monovalent residue remaining after removal of onehydrogen atom from an aromatic or heteroaromatic compound which canconsist of one ring or two fused or catenated rings having 5 to 12 ringatoms which can include up to 3 heteroatoms selected from S, N, andnonperoxidic O. The carbon atoms can be substituted by up to threehalogen atoms, C₁ -C₄ alkyl, C₁ -C₄ alkoxy, N,N-di(C₁ -C₄ alkyl)amino,nitro, cyano, and C₁ -C₄ alkyl carboxylic ester;

"arenyl" means the monovalent residue remaining after removal of ahydrogen atom from the alkyl portion of a hydrocarbon containing bothalkyl and aryl groups having 6 to 26 carbon and heteroatoms (wherein theheteroatoms are up to 3 S, N, and nonperoxidic O atoms);

"azlactone" means 2-oxazolin-5-one groups of Formula I and2-oxazin-6-one groups of Formula II; ##STR3## "parts" means parts byweight unless otherwise specified; "carboxylate" means ##STR4## whereinM is hydrogen, ammonium, or an alkali metal such as Li, Na, or K;"macroporous" refers to crosslinked polymers in which the level ofcrosslinker or difunctional monomers is greater than 20 parts, with nopolymer non-solvent or porogen utilization being required;

"biologically active" refers to substances which are biochemically,immunochemically, physiologically or pharmaceutically active such asantibodies, antigenic substances, enzymes, cofactors, inhibitors,lectins, hormones, receptors, coagulation factors, amino acids,histones, vitamins, drugs, cell surface markers, and substances whichinteract with them;

"gel-type" refers to crosslinked polymers in which the level ofcrosslinkers or difunctional monomers is less than 20 parts.

Structures and formulae depicted between parentheses are partialstructures of polymers.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides azlactone-functional supports having onat least one of their surfaces units of Formula V: ##STR5## wherein R1¹is H or CH₃,

R² and R³ independently can be an alkyl group having 1 to 14 carbonatoms, a cycloalkyl group having 3 to 14 carbon atoms, an aryl grouphaving 5 to 12 ring atoms, an arenyl group having 6 to 26 carbon and 0to 3 S, N, and nonperoxidic O heteroatoms, or R² and R³ taken togetherwith the carbon to which they are joined can form a carbocyclic ringcontaining 4 to 12 ring atoms, and

n is an integer 0 or 1.

These supports may be crosslinked azlactone- functional polymeric beadsor they may be solid substrates coated on at least one surface with alayer of an azlactone-functional polymer. This layer may have athickness in the range of 1 nanometer to 5 mm. Useful solid substratesinclude inorganic solids such as glass, ceramics, unfired metal andnonmetal oxides, clays, zeolites, and organic polymers.

Also provided by this invention are adduct supports having the formula##STR6## wherein R¹, R², R³, and n are as previously defined,

X can be --O--, --S--, --NH--, or ##STR7## wherein R⁴ can be alkyl oraryl, and G is the residue of HXG which performs the adsorbing,complexing, catalyzing, separating, or reagent function of the adductbeads.

HXG can be a biologically active substance, dye, catalyst, reagent, andthe like.

The azlactone-functional supports of this invention are provided by oneof several processes:

PROCESS I Two-step Reverse Phase Suspension Polymerization

The polymer and adduct supports of Process I of the invention can beprovided according to the process depicted by Chemical Equations I,below. ##STR8##

The crosslinked hydrophilic, azlactone-functional polymer beads ofFormula V are prepared by a novel two-step process. In the first stepthe following group of monomers is subjected to a free radicalpolymerization reaction:

i) 0 to 89 molar parts of at least one water soluble monomer;

ii) 1 to 99.9 molar parts of at least one water soluble salt ofN-(meth)acryloylamino acid; and

iii) in the range of 0.1 to 99 molar parts, preferably 7 to 99, morepreferably 10 to 99, and most preferably 30 to 99 molar parts, of atleast one crosslinking monomer.

The product of the above polymerization reaction is the crosslinked,hydrophilic, carboxylate-functional supports of Formula IV. The secondstep of the process involves treating the carboxylate-functionalsupports with a cyclization agent to form the azlactone-functionalsupports of the invention.

The degree of hydrophilicity of the polymer support is largelydetermined by the amount of water soluble monomer employed, althoughsome hydrophilicity is imparted by the crosslinker and by the functionalgroups created, i.e., amide-amide, amide-ester, or amide-thiolester withamine, alcohol, or thiol nucleophiles (HXG as defined above), by thering-opening, azlactone/nucleophile reaction (step 3 of ChemicalEquations I). Therefore, in the strictest sense of the presentinvention, inclusion of a water soluble monomer is optional. Suitablewater soluble monomers exhibit a solubility of at least 3 parts in 100parts water. Preferred monomers include vinyl group-containing andacryloyl group-containing compounds. A representative list of suchmonomers includes acrylamide, methacrylamide, N,N-dimethylacrylamide,diacetoneacrylamide, N-vinylpyrrolidone, hydroxyethyl methacrylate,2-acrylamido-2-methylpropanesulfonic acid and its salts,N-(3-methacrylamidopropyl)-N,N,N-trimethylammonium salts,N,N-dimethylaminoethyl methacrylate, acrylic acid, methacrylic acid,itaconic acid, and combinations thereof. Preferred water solublemonomers are N,N-dimethylacrylamide and N-vinylpyrrolidone.

The N-acryloylamino acid salt monomers include ammonium, sodium,potassium, and lithium salts of N-acryloylamino acids of Formula VII andare prepared by mixing (at <30° C.) equal molar quantities of aqueoussolutions of, for example, ammonium hydroxide, sodium hydroxide,potassium hydroxide, or lithium hydroxide and the Formula VII compounds.##STR9## wherein R¹, R², R³, and n are as previously defined. TheN-acryloylamino acid compounds are well-known and can be readilysynthesized. For Formula VII compounds in which n=0, either the sodiumsalt of the appropriate amino acid can be acryloylated, for example,according to K. Huebner, et al., Makromol. Chem., 11, 109 (1970) or,more efficiently, by the method described in U.S. Pat. No. 4,694,103which involves the one-pot transformation of a ketone into anN-acryloylamino acid. For Formula VII compounds wherein n=1, a usefulpreparation is the transformation of 3,3-disubstituted acrylic acids asdisclosed by D. I. Hoke, et al., J. Polym. Sci.: Polym. Chem. Ed., 10,3311 (1972).

Insolubilization is a necessary condition for easy removal of thesupport (e.g., beads) from the system. This is accomplished by inclusionof a monomer which contains a plurality of polymerizable groups andwhose participation in a polymerization reaction results in the physicaljoining of polymer backbones or crosslinking. Crosslinking is alsodesirable in polymer-supported materials because the mechanicalstability is generally substantially enhanced and some degree of controlof bead size can be exercized by manipulation of the level ofcrosslinking, i.e., in general for a given polymerization condition, thegreater the amount of crosslinker the smaller the bead size. The degreeof crosslinking depends primarily on the intended use of the supportmaterial. In all instances the polymers are insoluble in all solventsand possess a molecular weight which is essentially infinite. For manyapplications requiring fairly high capacities and involving relativelysmall solute reaction partners which can diffuse into the swollenpolymer support, low to moderate degrees of crosslinking are desired.According to D. C. Sherrington, Br. Polym. J., 16, 164 (1984), thesecrosslinked swellable supports (referred to as "gel-type" polymers)result from inclusion of from 1 to 20 parts of a multifunctionalmonomer. For certain applications requiring low degrees of physicalexpansion due to swelling and which can tolerate lower capacities, (asin certain operations conducted in confined flow systems such aschromatographic columns or column reactors), highly crosslinkedhydrophobic systems resulting from copolymerization of more than 20parts of a multifunctional monomer are utilized. These are so-called"macroporous" polymers which are generally regarded as beingnon-swelling, and solute/support reactions occur primarily at thesolvent/support interface. Applications of these supports may involvelarge solutes, e.g., biomacromolecules, which cannot, because of theirlarge size, diffuse into the polymer network.

In sum, the prior art teaches that in hydrophobic systems 20 parts ormore of crosslinker results in a non-swelling system.

We have found with the hydrophilic supports of the present invention,however, that in order to achieve a condition of low swelling in aqueousmedia, a substantially greater concentration of multifunctional monomeris necessary than the 20 parts commonly utilized in the so-callednon-swelling, hydrophobic, macroporous resins described above. This maybe a consequence of the utilization of these hydrophilic supports inwater and the high degree of hydrophilicity imparted by themultifunctional monomers themselves, as they consist largely of highlypolar functional groups.

The prior art generally has taught polymer supports (beads) comprisinghydrophobic comonomers and hydrophobic crosslinking monomers in order toachieve crosslinked polymer beads. These were known to be swellable when1 to 20 parts of crosslinker were present. Above 20 parts ofdifunctional monomer (crosslinker) provided essentially non-swellingbeads. U.S. Pat. No. 4,070,348 teaches that 0.2 to 5 mol % ofcrosslinking monomer provides beads with a high degree of swelling inwater. The patentee believes that this low degree of crosslinking andaccompanying high degree of swelling is necessary to achieve highbinding capacity.

In the instant invention, hydrophilic comonomers and hydrophiliccrosslinkers are utilized. Swelling of beads so produced variesinversely with the amount of multifunctional crosslinker present.Polymer supports (e.g., beads packed together) with a low degree ofswelling (less than 3 times the unswelled volume) generally requiresubstantially greater than 20 parts of difunctional crosslinker.

Surprisingly, there can still be a relatively low degree of swelling andhigh binding capacities of polymer beads in water with more than 5 mol %crosslinker (in hydrophilic systems). Such beads are useful ascomplexing agents, catalysts, polymeric reagents, chromatographicsupports, and enzyme-, other protein-, and otherbiomacromolecule-bearing supports.

To achieve polymer beads with a low degree of swelling and stillmaintain high binding capacity, substantially greater amounts ofcrosslinker are required in hydrophilic systems. Such polymer beads areparticularly useful in chromatographic applications and column reactors.

Suitable multifunctional crosslinking monomers include ethylenicallyunsaturated (α,β-unsaturated) esters such as ethylene diacrylate,ethylene dimethacrylate, trimethylolpropane triacrylate andtrimethacrylate, and α,β-unsaturated amides, such asmethylenebis(acrylamide), methylenebis(methacrylamide),N,N'-diacryloyl-1,2-diaminoethane,N,N'-dimethacryloyl-1,2-diaminoethane, and reaction products of2-alkenyl azlactones and short chain diamines such as those representedby Formulae VIII and IX: ##STR10## The crosslinking monomers should beat least sparingly soluble in water but need not be as water soluble asdefined for the water soluble monomer component. This is not generally aproblem for the preparation of gel-type polymers because relativelysmall proportions of the crosslinking monomers are utilized withrelatively large quantities of water solvent, and often the watersoluble monomer component, especially N,N-dimethylacrylamide andN-vinylpyrrolidone, will facilitate solution of the crosslinkingmonomer. For macroporous polymers, however, in which the concentrationof crosslinking monomer is greater than 20 parts it may be necessary toadd a co-solvent which will facilitate dissolution of the crosslinkingmonomer. Suitable co-solvents include N,N-dimethylformamide,N,N-dimethylacetamide, N-methylpyrrolidone, and dimethylsulfoxide.

The technique of polymerization employed in the present inventionPROCESS I is often referred to as "reverse-phase" or "inverse"suspension polymerization, and a general discussion of this technique isdisclosed by M. Munzer, et al., "Suspension Polymerizations fromNon-Aqueous Media", in "Polymerization Processes" edited by C. E.Schildknecht and I. Skeist, Wiley-Interscience, New York, pp. 123-124(1977). The reversal of the normal suspension polymerization technique(in which water is the usual suspending medium) is necessary because themonomers of the present invention are soluble in water and thereforerequire a water immiscible suspending medium.

The primary purpose of the suspending medium, besides functioning as aninert medium for dispersion of the polymerizable phase, is to dissipatethe heat generated in the polymerization reaction. An importantcharacteristic of the suspending medium is its density. In order toobtain spherical polymer beads of uniform size, the beads, once formed,should not exhibit a tendency to sink or float in the suspending medium.Therefore, the suspending medium and aqueous phases should be ofapproximately the same density.

The actual polymerization occurs in individual droplets of watercontaining the dissolved monomers and initiator. The droplets are formedand maintained in the suspending medium by vigorous agitation, and theresultant beads' size and individuality (i.e., lack of aggregation) arecontrolled by the addition of various suspending agents which aresurface active molecules that generally contain both hydrophobic andhydrophilic parts.

In and of itself, the polymerization step (step one) is not a novelaspect of the present invention. As is apparent to one skilled in theart, the nature of the suspending medium, the amount of water employed,the initiation system, the amount of crosslinking agent, the stirringrate, and the suspending agent are all essentially independent andimportant variables that determine the shape and size of the polymericbeads. While not wishing to be bound by any particular set ofpolymerization conditions, we have found the reverse-phase suspensionpolymerization procedure described by G. L. Stahl, et al., J. Org. Chem,44, 3424 (1979) to be exceedingly useful. In that procedure a mixture ofheptane and carbon tetrachloride is utilized as the suspending medium;the initiation system is the ammoniumpersulfate/N,N,N',N'-tetramethyl-1,2-diaminoethane redox couple; thestirring rate is 300 rpm; and the suspending agent is sorbitansesquioleate. Substitution of the various components by comparablematerials can certainly be made, and such substitutions would not beoutside the spirit and scope of the present invention. For example,utilizing a polymeric stabilizer such as copoly(isooctylacrylate/acrylicacid) or copoly(hexylacrylate/sodium acrylate) instead of sorbitansesquioleate was found to provide more consistently nonaggregated beadproducts.

Step two of PROCESS I of the invention consists of conversion of thecarboxylate-functional beads into azlactone-functional beads. This isaccomplished using a cyclization agent (CA). A cyclization agent is areagent that can react with the carboxylate-functional beads to form anintermediate adduct which is susceptible to intramolecular attack by theamide carbonyl group to form azlactone groups according to CHEMICALEQUATIONS IA. This susceptibility is chiefly accomplished by forming agood leaving group (⁻ O(CA) below) for the nucleophilic attack by thecarbonyl. ##STR11## wherein R¹, R², R³, and n are as defined above.

(Structures and formulae depicted between parentheses are partialstructures of polymers depicting side chains that actively participatein the cyclization reaction. Use of brackets has the usual meaning ofchemical intermediates or activated complexes. Dotted lines mean partialbonds, and δ means partial ionic charges.)

Useful cyclization agents for transformation of thecarboxylate-functional supports include, by way of example, aceticanhydride, trifluoroacetic anhydride, and alkyl chloroformates such asmethyl, ethyl, and isopropyl chloroformates. Carbodiimides such asN,N'-dicyclohexylcarbodiimide can be effectively utilized but require anadditional step of acidifying the carboxylate-functional supports toform carboxyl-functional supports which can then be cyclized toazlactone-functional supports using the carbodiimide reagent. Tofacilitate understanding of the cyclization step of the invention, theintermediates that would result by employing the aforementionedcyclization agents are depicted below in order of mention. ##STR12##

The progress of the cyclization reaction can be easily monitored byexamination of the infrared spectrum of the polymer supports. Appearanceof a carbonyl stretching absorption at about 1820 cm⁻¹ is evidence ofazlactone groups. Indeed, one reason azlactone groups are so useful aslinkages for covalent attachment to polymers is the ability to monitorreactions by observation of this infrared absorption, either theappearance of it in the synthesis of the azlactone-functional supportsor the disappearance of it in the subsequent reaction with a functionalmaterial. This absorption is strong, very characteristic of azlactones,and located in a region of the infrared spectrum where essentially noother common absorptions are observed. This is a decided advantage overother linking functional groups such as the chloromethylphenyl andoxirane which lack these unique features in their infrared spectra. Aconvenient analytical method for monitoring attaching reactions reallydoes not exist with these latter groups.

Because of its low cost, availability, and liquid state at cyclizationtemperatures, acetic anhydride is a preferred cyclization agent.Typically, the carboxylate-functional supports are covered with aceticanhydride, and the mixture is warmed at temperatures from 40°-100° C.,preferably 80°-100° C., for a period of 2-24 hours. After thecyclization reaction, the polymer supports are filtered. What also makesacetic anhydride particularly preferred is that the by-product ofcyclization, the alkali metal acetate salt, is fairly soluble in aceticanhydride and can easily be removed from the azlactone-functionalsupports. The supports can then be dried directly or, as is oftenconducted, subjected to a series of washing operations with non-reactiveorganic solvents such as acetone, toluene, ethyl acetate, heptane, andchloroform prior to drying.

PROCESS II One-Step Reverse Phase Suspension Polymerization

Polymeric supports of PROCESS II of the invention are provided accordingto the process depicted in CHEMICAL EQUATIONS II, below. ##STR13##

This process is conducted by the same polymerization technique as thatemployed in PROCESS I, and employs the same water soluble monomers andcrosslinkers. The major difference is in the utilization of an alkenylazlactone monomer X instead of the N-acryloylamino acid salt III. Theamounts of reactants can be the same as for PROCESS I except thatazlactone replaces the salt of N-(meth)acryloylamino acid. This processadvantageously provides azlactone-functional polymer supports V in asingle step, as opposed to the two-step process of PROCESS I. Severalaspects of this process are surprising in light of the prior art. Firstof all, the alkenyl azlactones X are fairly soluble in the suspendingmedium, yet they become readily incorporated in the polymer support(e.g., beads) without detrimental effects upon the polymerizationprocess. (This is in sharp contrast to what is observed employing theteachings of U.S. Pat. No. 4,070,348 (See Examples 47 and 48 below.))Secondly, the azlactone ring is not hydrolyzed by the water in theaqueous phase during this polymerization process. This is alsoremarkable considering the teachings of U.S. Pat. No. 4,070,348 and ofTaylor, supra. After the polymerization process, the beads can beisolated, for example, by filtration, and subjected to a series ofwashing steps, if desired, and dried.

Useful azlactone monomers and their syntheses are described in U.S. Pat.No. 4,378,411 and in "Polyazlactones", Encyclopedia of Polymer Scienceand Engineering, Vol. 11, Second Edition, Wiley, N.Y., 1988, pp.558-571, both of which are incorporated herein by reference, andinclude:

2-vinyl-4,4-dimethyl-2-oxazolin-5-one,

2-isopropenyl-4,4-dimethyl-2-oxazolin-5-one,

2-vinyl-4,4-diethyl-2-oxazolin-5-one,

2-vinyl-4-ethyl-4-methyl-2-oxazolin-5-one,

2-vinyl-4-dodecyl-4-methyl-2-oxazolin-5-one,

2-vinyl-4,4-pentamethylene-2-oxazolin-5-one,

2-vinyl-4-methyl-4-phenyl-2-oxazolin-5-one,

2-isopropenyl-4-benzyl-4-methyl-2-oxazolin-5-one, and

2-vinyl-4,4-dimethyl-1,3-oxazin-6-one.

Preferred azlactone monomers are

2-vinyl-4,4-dimethyl-2-oxazolin-5-one (which is commercially availablefrom SNPE, Inc., Princeton, N.J.),

2-isopropenyl-4,4-dimethyl-2-oxazolin-5-one, and

2-vinyl-4,4-dimethyl-1,3-oxazin-6-one.

PROCESS III Dispersion Polymerization

Polymeric supports of PROCESS III of the invention are provided by apolymerization process termed "dispersion polymerization", and inparticular, by dispersion polymerization in organic media. In thisprocess which is somewhat analogous to PROCESS II, the monomers andsolvent are initially homogeneous. Shortly after polymerization begins,polymer separates as particles and the polymerization then continues ina heterogeneous manner. Polymeric "dispersants" or "stabilizers" aretypically used to prevent aggregation of polymer particles during thepolymerization process. Techniques for dispersion polymerization innon-aqueous media are well-known in the art, and are described indetail, for example, by K. E. J. Barrett in "Dispersion Polymerizationin Organic Media", Wiley, N.Y., 1975. A dispersion polymerizationtechnique which has proven advantageous for the preparation ofazlactone-functional supports of the present invention of PROCESS III isthat described by Y. Almog, et al., Brit. Polym. J., 1982, 131, which isincorporated herein by reference.

In general, azlactone-functional polymer supports of Formula V areprepared according to PROCESS III by subjecting to a free radicalpolymerization reaction the following group of monomers:

i) 1-100 molar parts of at least one alkenyl azlactone of Formula X;

ii) 0-99 molar parts of at least one crosslinking monomer; and

iii) 0-99 molar parts of at least one comonomer.

Suitable crosslinking monomers for use in this polymerization processinclude the ones useful for PROCESSES I and II. However, since watersolubility is not a criterion in dispersion polymerization but rathersolubility in the dispersing medium, other crosslinkers may be utilizedsuch as, for example, divinyl compounds such as divinylbenzene.

Comonomers useful for the preparation of supports according to PROCESSIII include the water soluble comonomers useful in PROCESSES I and II,but again include additional comonomers which are not water soluble.Virtually any free radically polymerizable monomer may be utilized ascomonomer subject to the requirement that it have initial solubility inthe dispersing medium.

Examples include: the vinyl aromatic monomers such as styrene,α-methylstyrene, 2- and 4-vinylpyridine; α,β-unsaturated carboxylicacids such as acrylic acid, methacrylic acid, itaconic acid, maleicacid, fumaric acid, and crotonic acid; α,β-unsaturated carboxylic acidderivatives such as methyl methacrylate, butyl methacrylate,2-ethylhexyl methacrylate, ethyl acrylate, butyl acrylate, iso-octylacrylate, octadecyl acrylate, cyclohexyl acrylate, tetrahydrofurfurylmethacrylate, phenyl acrylate, phenethyl acrylate, benzyl methacrylate,α-cyanoethyl acrylate, maleic anhydride, diethyl itaconate, acrylamide,methacrylonitrile, N,N-dimethylacrylamide, and N-butylacrylamide; vinylesters of carboxylic acids such as vinyl acetate and vinyl2-ethylhexanoate; vinyl halides such as vinyl chloride and vinylidenechloride; vinyl alkyl ethers such as methyl vinyl ether, 2-ethylhexylvinyl ether, and butyl vinyl ether; olefins such as ethylene; N-vinylcompounds such as N-vinylpyrrolidone and N-vinylcarbazole; vinyl ketonessuch as methyl vinyl ketone; and vinyl aldehydes such as acrolein andmethacrolein.

As is well known to one skilled in the art of dispersion polymerization,an inert diluent or dispersing medium must be chosen which will dissolvethe monomer or monomer mixture but will precipitate the polymer as itforms. This presents a particular problem when preparing crosslinkedpolymers, since they are insoluble in all solvents. Therefore adispersing medium must be chosen which will favor the separation ofdiscrete particles during the polymerization process rather thanformation of a crosslinked mass. A useful concept to aid in thedetermination of dispersing media or in choosing appropriate monomermixtures which may be dispersion polymerized in a particular medium isthe concept of solubility parameter. This concept and its relationshipto dispersion polymerization is discussed in detail by Barrett, supra(Chapter 4). Tables of solubility parameter values for many solvents andsome polymers, as well as methods for the estimation of solubilityparameter values for polymers and copolymers, can be found in PolymerHandbook, J. Brandrup and E. H. Immergut, Editors, 2nd Edition, Wiley,New York, 1975, p. IV-337ff. In general, for a successful dispersionpolymerization, the solubility parameter of the dispersing medium and ofthe polymer being formed should differ by at least about 1 to 1.5solubility parameter units, preferably by 1.5 to 2 or more solubilityparameter units. Therefore, for most monomer mixtures, solvents usefulas dispersing media include nonpolar hydrocarbons such as pentane,hexane, petroleum ether, cyclohexane, and toluene, and the polar,hydroxylic solvents such as the alcohols methanol, ethanol, isopropanol,and t-butanol.

Initiators useful for PROCESS III of the invention include all freeradical initiators which are soluble in the dispersing medium. Choice ofthe initiator will depend, as is well known in the art, upon thetemperature at which the polymerization is conducted. Initiators usefulat elevated temperatures, such as at 50° C. or higher, include azocompounds, such as azobisisobutyronitrile, and peroxides orhydroperoxides such as benzoylperoxide, di-t-butylperoxide,t-butylhydroperoxide, and cumene hydroperoxide. For lower temperaturereactions, for example at room temperature, redox initiators may beutilized such as, for example, peroxides or hydroperoxides incombination with a tertiary amine. One such redox system is benzoylperoxide/N,N-dimethylaniline. Initiators can be present in an amount inthe range of 0.1 to 10 weight percent of the monomer composition,preferably 0.5 to 2.0 weight percent.

As mentioned above, the dispersion polymerization procedure of Almog, etal., has been used effectively for the preparation ofazlactone-functional supports by PROCESS III. This procedure employs analcohol as the dispersing medium, and azobisisobutyronitrile as theinitiator. A polymeric stabilizer such as polyvinylpyrrolidone,poly(vinyl methyl ether), polyacrylic acid, or polyethyleneimine is usedin conjunction with Aliquat 336 (Henkel Corporation) as cosurfactant.Again a surprising and unexpected result of this procedure is thatazlactone-functional polymer supports, both crosslinked andnoncrosslinked, may be prepared in one step in this hydroxylic mediumwithout reaction of the alcohol solvent with the azlactone. Isolationinvolves a simple filtration, washing if desired, and drying.

While the beads prepared by the three processes described above allexhibit azlactone functionality on their surfaces, their physicalproperties may vary widely depending upon the process used for theirpreparation. The beads prepared via reverse phase suspensionpolymerizations are generally highly porous (i.e., 10 to 90 volumepercent voids, preferably 20 to 75 volume percent voids), with largesurface areas and pore volumes, and have a high density of reactivegroups. These beads are useful for applications in which bindingcapacities are of relatively more importance than are reaction kinetics.Beads produced by dispersion polymerizations, on the other hand, aregenerally smaller in size and are much less porous, in some instancesbeing virtually nonporous. With these beads, reaction kinetics are veryfast, a characteristic which can be particularly useful in certainapplications such as those requiring higher throughput rates.

PROCESS IV Coating Solid Supports With Uncrosslinked Azlactone Polymers

As noted above, polymeric supports of the invention can be in the formof beads. This is a physical form in which the supports possess greatutility, particularly for uses such as packing chromatographic columns.However, the new materials are not restricted to the physical form ofbeads. We have found that certain soluble azlactone polymers(uncrosslinked) can also be coated on a number of substrates and theyexhibit the same reactive azlactone functionality in these forms as theydo as beads. Thus, these substrates may be used for reaction withfunctional materials. For example, nylon filtration membranes and glasssurfaces can be coated with azlactone polymers of this invention, bydipping the object to be coated into a solution of the polymer andallowing the dipped object to dry. Similarly, particulate material, suchas ceramics (e.g., zirconium oxide) or unreactive polymers, such asparticles of polyethylene, can be coated with azlactone functionalpolymers. Other solution coating methods well known in the art may beused, such as for example spray coating and knife coating, dependingupon the physical form of the substrate.

Similar results have been obtained when silica beads were used as asubstrate upon which azlactone-functional polymers of this inventionwere coated. This kind of bead is commonly used as a packing inchromatographic columns. Likewise, using glass beads of controlled poresize, again a common column packing medium, significantly improvedprotein binding and covalent binding have been found when using anazlactone-functional coating. Particular advantages ofazlactone-functional supports of PROCESS IV are their incompressibilityand almost complete lack of swelling.

The azlactone functional polymers useful for preparing coatings on solidsubstrates are well known in the art or can be prepared by techniqueswell known in the art. These polymers are prepared in general by freeradical polymerization of one or more alkenyl azlactones, optionallywith one or more free radically polymerizable, ethylenically unsaturatedcomonomers, using polymerization procedures common in the art. Suitableazlactone containing polymers and copolymers are described, for example,in R. Huebner, et al., Angew. Makromol. Chem., 1970, 11, 109 and in U.S.Pat. No. 4,378,411. Particularly suitable azlactone-functional polymersfor preparing coatings on solid supports can be prepared by reacting aportion of the azlactone groups of the above azlactone-containinghomopolymers or copolymers with a lower alkyl amine or alcohol.

Other methods are available for preparing azlactone-functional supports.One method is to apply alkenyl azlactone monomer to the support(optionally along with other co-monomers) and polymerize the monomer(s)in place. Methods of polymerization include photopolymerization(utilizing an appropriate photoinitiator) as is well known in the art.

The azlactone-functional polymer supports of the invention have now beenformed and are ready for reaction with a functional material. Asindicated earlier, a surprising discovery was that functional materialscan often be attached to azlactone-functional supports of the inventionin solvents such as water that have heretofore been thought of as beingreactive with azlactones. "Material" as used herein means the principalchemical entity that is desired to be attached to a polymer support toaccomplish a specific purpose. Stated another way, "material" means thatportion or residue of the "functional material" which actually performsthe adsorbing, complexing, catalytic, or reagent end-use. "Functional"for purposes of this invention means that portion of the "functionalmaterial" which contains a group that can react with an azlactone."Functional" groups useful in the present invention include hydroxy,primary amine, secondary amine, and thiol. These groups react, either inthe presence or absence of suitable catalysts, with azlactones bynucleophilic addition as depicted in equation (2) below. ##STR14##wherein R¹, R², R³, n, X, and G are as previously defined.

Depending on the functional group present in the functional material,catalysts may be required to achieve effective attaching reaction rates.Primary amine functional groups require no catalysts. Acid catalystssuch as trifluoroacetic acid, ethanesulfonic acid, toluenesulfonic acid,and the like are effective with hydroxy and secondary amine functionalgroups. Amine bases such as triethylamine,1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) and1,5-diazabicyclo[4.3.0]non-5-ene (DBN) (both availabale from AldrichChemical Co., Milwaukee, Wis.) are effective as well for hydroxy andthiol functional groups. The level of catalyst employed is generallyfrom 1 to 10 parts, preferably 1 to 5 parts, based on 100 parts ofazlactone.

As is apparent to one skilled in the art, specific reaction conditionssuch as solvent, temperature, level of catalyst, etc. vary tremendouslydepending on the functional material that is to be attached. Because ofthe myriad of functional materials that have been or could be attachedto polymer supports, any listing of functional materials beyond thegeneric HXG of equation (2) and CHEMICAL EQUATIONS I would be incompleteand somewhat unnecessary, as the inventive aspects of the presentinvention do not reside with the functional materials. What is novel isthat these functional materials can be covalently bound to azlactonefunctional supports.

Having described the invention in general terms, objects and advantagesof the invention are more specifically illustrated by the followingexamples. The particular materials and amounts thereof recited in theexamples, as well as other conditions and details, should not beconstrued to unduly limit this invention. In the Examples below numbersin parentheses ( ) are in weight percent, and those in brackets [ ] arein mole percent.

EXAMPLE 1

This example teaches the preparation of an azlactone functional supportaccording to PROCESS I.

Preparation of Copoly(N,N-Dimethylacrylamide:2-Vinyl-4,4-Dimethylazlactone:Methylenebisacrylamide) (46:46:8)[54.8:39.0:6.1]

Step 1: Preparation of Copoly(N,N-Dimethylacrylamide (DMA):N-Acryloylmethylalanine Sodium Salt (NaAMA):Methylenebisacrylamide(MBA)) (44.6:50.4:7.8): A two-liter creased, round bottomed flaskequipped with a mechanical stirrer (stirring rate about 300 rpm),nitrogen inlet, thermometer, and condenser was charged with heptane(1043 mL) and carbon tetrachloride (565 mL). This solution was stirredand sparged with nitrogen for 15 minutes. A separate solution wasprepared consisting of a sodium hydroxide solution (6.6 grams; 0.165mole dissolved in 85 mL of water), N-acryloylmethylalanine (AMA) (25.98grams; 0.165 mole), DMA (23 grams; 0.232 mole), MBA (4 grams; 0.026mole), and ammonium persulfate (1 gram; 0.004 mole) and added to theorganic suspending medium. Sorbitan sesquioleate (Arlacel™ 83, ICIAmericas, Inc., Wilmington, Del.) (2 mL) was added and the mixturestirred and sparged with nitrogen for 15 minutes.N,N,N',N'-tetramethyl-1,2-diaminoethane (2 mL) was added and thereaction temperature rose fairly quickly from 21° C. to 33° C. Themixture was stirred at room temperature for three hours. The mixture wasefficiently filtered using a "D" (greater than 21 micrometers) sinteredglass funnel, and the filter cake washed thoroughly and repeatedly withacetone. After drying at 60° C. and less than 1 Torr. for 12 hours, thedry solid (52 grams) was sieved and separated into four fractions: beadsless than 38 micrometers, 12.32 grams; beads between 38 and 63micrometers, 19.83 grams; beads between 63 and 90 micrometers, 4.56grams; and beads greater than 90 micrometers, 13.95 grams. Employing anoptical microscope arrangement consisting of a Nikon NomarskiDifferential Interference Contrast Microscope, a Dage Newvicon videocamera, a Sony 3/4" video recorder, and a Perceptive Systems, Inc.digital image processor with accompanying software, it was determinedthat the 38-63 micrometer sample consisted of quite spherical beads(average aspect ratio=0.87) which swell in water with an accompanyingincrease in diameter of from 35-50%.

Step 2: Cyclization to Copoly(DMA:2-vinyl-4,4-dimethylazlactone(VDM):MBA) (46:46:8): Acetic anhydride (100 mL) was added to 15.1 gramsof the 38-63 micrometer beads prepared in Step 1. The mixture was heatedto 100° C. for two hours. After cooling and filtering, the beads wereplaced in a Soxhlet extraction apparatus and were extracted with ethylacetate for 16 hours. After drying at 60° C. and less than 1 Torr., thebeads weighed 12.6 grams.

EXAMPLE 2

This example teaches use of the reaction product of 1,2-diaminoethaneand VDM as a crosslinking monomer (PROCESS I).

Preparation ofN,N'-bis(2-acrylamido-2-methylpropionyl)-1,2-diaminoethane

A 100 mL, three-necked, round bottomed flask equipped with a magneticstirring bar, a dropping funnel, thermometer, and condenser was chargedwith VDM (13.9 grams; 0.10 mole) and tetrahydrofuran (50 mL). A solutionof 1,2-diaminoethane (3.0 grams; 0.05 mole) in tetrahydrofuran (10 mL)was added dropwise such that the temperature did not exceed 30° C. Afterstirring overnight the reaction mixture was filtered to remove a whitesolid which after washing with hexane and drying at less than 1 Torr.weighed 15.8 grams (93% yield). The solid melted at 207°-210° C. andexhibited satisfactory elemental analyses and spectral characteristicsfor the desired material, which is the compound of Formula VIII in thespecification.

Preparation ofCopoly(N,N-Dimethylacrylamide:2-Vinyl-4,4-Dimethylazlactone:N,N'-Bis(2-Acrylamido-2-Methyl-Propionyl)-1,2-Diaminoethane)(55:42.7:2.3)

The two-step procedure of Example 1 was utilized except MBA was replacedby the above prepared crosslinking monomer (1.0 gram; 0.003 mole). Asample (15.1 grams) of the intermediate carboxylate-functional polymerwas treated with acetic anhydride to yield, after washing and drying,11.0 grams of the azlactone-functional polymer.

EXAMPLE 3

This example teaches the reaction of a gel-type polymer and a relativelylow molecular weight, intrapolymer support-diffusible functionalmaterial. The example further teaches a procedure for quantitativedetermination of azlactone groups.

The procedure is a variation of a quantitative analysis of isocyanatesand isothiocyanates using n-butylamine (cf. S. Siggia, "QuantitativeOrganic Analysis via Functional Groups", John Wiley & Sons: New York, p.558 (1963)). Generally, the procedure involves treatment of theazlactone-functional beads with standard triethylamine inN,N-dimethylformamide (DMF) to react with and determine theconcentration of any uncyclized carboxyl groups. To another sample ofbeads, excess standard n-butylamine in DMF is added and shaken for 24hours at room temperature. The excess concentration of n-butylamine isthen determined by potentiometric titration with standard acid as anindirect measure of the concentration of azlactone groups. Using thismethod with the beads of EXAMPLE 2, three separate determinations showedminimal, i.e., less than 0.3 milliequivalents/gram (meq/g) of resin,carboxyl content and an average azlactone content of 2.2 meq/g.Theoretical azlactone content was 3.1 meq/g. Therefore, over 70% of thetheoretical azlactone groups had formed and were accessible by then-butylamine functional material.

EXAMPLE 4

This example further teaches the reaction of a gel-type polymer with arelatively small functional material, N-(3-aminopropyl)morpholine, butin an aqueous reaction solvent. Determination of reactable azlactonecontent is made by measuring the increase in % nitrogen of the reactedbeads. This procedure is more time consuming than the quantitativeanalysis method outlined in EXAMPLE 3, but comparison of the resultsserves as a check on the accuracy of the titration method.

A gel-type polymer consisting of DMA:VDM:MBA (53.8:41.7:4.5)[62.3:34.4:3.3] was prepared as in EXAMPLE 1; the theoretical % nitrogenpresent in the beads should be 12.6%; experimentally observed using aKjeldahl method was 12.1%.

The azlactone-functional beads (1.44 grams; containing approximately0.004 mole of azlactone groups), N-(3-aminopropyl)morpholine (0.80 gram;0.0055 mole), and 15 mL of a standard aqueous pH 9 buffer solution wereplaced in a 100 mL, round bottomed flask and stirred at roomtemperature. After four hours the beads were filtered, washed repeatedlywith deionized water, and dried at 60° C. and less than 1 Torr. Theresulting adduct possessed a nitrogen content of 13.8%. Theoretically,the increase in nitrogen should have been 17.4%. The experimentallyobserved increase of 12.3% again indicates that 70% of the azlactonegroups had formed and reacted. This result is in excellent agreementwith the titration procedure result of EXAMPLE 3. Furthermore, theresult indicates that measurable hydrolysis in the aqueous pH 9 buffersolution did not occur and that virtually quantitative attachingreactions can take place in aqueous media at an elevated pH.

EXAMPLES 5-7

These examples illustrate how polymer bead size can be controlled by thelevel of crosslinking monomer (PROCESS I).

The procedure of Step 1 of EXAMPLE 1 was utilized to prepare thecarboxylate-functional beads of the following examples as shown in TABLEI, below. Average particle diameters were determined using an opticalmicroscope equipped with a Zeiss IBAS™ Image Analyzer. It is apparentthat as the level of crosslinker increases the particle diameterdecreases.

                  TABLE I                                                         ______________________________________                                                                      Average                                                Monomer wts. (g)                                                                             Wt %    particle                                               [mole %]       cross-  diameter                                        EXAMPLE  DMA     NaAMA    MBA   linker                                                                              (micrometers)                           ______________________________________                                        5        24      24       2     4     67.5                                           [62.2:34.4:3.3]                                                        6        23      23       4     8     42.2                                           [60.7:33.5:6.8]                                                        7        21      21       8     16    32.4                                            [55.6:30.7:13.6]                                                      ______________________________________                                    

EXAMPLES 8-10

These examples teach the preparation of highly crosslinked polymers ofthe invention (PROCESS I). They furthermore teach utilization of aco-solvent to facilitate dissolution of the crosslinking monomer.

The method of EXAMPLE 1 was utilized except the monomers and initiatorwere dissolved in water (75 grams) and DMF (30 grams). The azlactonecontent was determined utilizing the titration procedure of EXAMPLE 3,and is shown in TABLE II, below.

                                      TABLE II                                    __________________________________________________________________________                     Average                                                      Monomer wts. (g) particle                                                                              Azlactone content                                    [mole %]         diameter                                                                              (meq/g)                                              EX.                                                                              DMA NaAMA MBA (micrometers)                                                                         theoretical                                                                         measured                                       __________________________________________________________________________    8  34.02                                                                             4.48  12.5                                                                              26.0    0.5   0.29                                           [76.4:5.5:18.0]                                                               9  30.55                                                                             8.95  12.5                                                                              20.5    1.0   0.52                                           [70.1:11.4:18.4]                                                              10 27.08                                                                             13.42 12.5                                                                              25.0    1.5   1.10                                           [63.6:17.4:18.9]                                                              __________________________________________________________________________

The polymer beads prepared in Examples 8 to 10 can be reacted with afunctional material to provide a chromatographic support, a complexingagent, a polymeric reagent, or a catalyst.

EXAMPLE 11

This example teaches the preparation of a polymer withN-methacryloylmethylalanine sodium salt (NaMMA) instead of NaAMA(PROCESS I). The resulting azlactone-functional bead of Formula V wasformed with R¹ ═CH₃.

The procedure of EXAMPLE 9 was utilized except that NaMMA (9.65 grams)was substituted for the NaAMA. The resulting azlactone-functional beadswhich were formed after treatment with acetic anhydride had an averageparticle diameter of 22.4 micrometers and an azlactone functionality of0.68 meq/g.

EXAMPLE 12

This example teaches the preparation of a polymer withN-vinylpyrrolidone as the water soluble monomer component (PROCESS I).The procedure and monomer charges of EXAMPLE 9 were utilized except theDMA was replaced by N-vinylpyrrolidone. The average particle diameter ofthe beads resulting from Step 1 was 19.3 micrometers. Cyclizationafforded azlactone-functional beads which possessed a strong azlactonecarbonyl absorption band at about 1820 cm⁻¹ in the infrared.

EXAMPLE 13

This example teaches the synthesis of a six-membered ring azlactone(2-oxazin-6-one) functional polymer bead (PROCESS I).

The procedure of EXAMPLE 9 was utilized except3-acrylamido-3-methylbutyric acid sodium salt (9.65 grams) was utilizedinstead of NaAMA. After cyclization the 2-oxazin-6-one functional beadspossessed an average diameter of 28.5 micrometers and a functional levelof 0.16 meq/g.

EXAMPLE 14

This example teaches the reaction of an azlactone-functional polymerbead with a protein functional material.

Preparation of Radiolabeled Protein A

Protein A (2.5 mg) (from Staphylococcus aureus) (Genzyme Corp., Boston,Mass.) was dissolved in 10 mM potassium phosphate buffer (pH 7.0; 0.6mL) and two Iodo-beads™ (an insoluble form of chloramine T; PierceChemical Co., Rockford, Ill.) were added to catalyze the addition ofiodine to tyrosine residues. The reaction was initiated by the additionof 0.1 milli Curies (mCi) of NaI (carrier-free ¹²⁵ I, New EnglandNuclear Co., N. Billerica, Mass.). The reaction was incubated at 20° C.for 30 minutes with vigorous manual shaking at five minute intervals.Protein A (both iodinated and unmodified forms) was separated from NaIby elution through a Pharmacia PD-10 size exclusion column in the samephosphate buffer. The fractions which contained protein were combined,aliquotted, and frozen at -15° C. until used. Specific radioactivity onday 0 was 154,000 counts per minute (cpm)/μg. All subsequentcalculations were corrected for the radioactive half-life of ¹²⁵ I of 60days. Radioactive Protein A was not used beyond six weeks afteriodination.

Reaction of the Radiolabeled Protein A with an Azlactone-Functional Bead

The azlactone-functional polymer utilized was that prepared in EXAMPLE5. The polymer beads (0.010 gram) were placed in a centrifuge tube andwere covered with a solution consisting of the labeled Protein Apreparation above (100 μL) and 400 μL of a phosphate buffer solution (pH7.5). The mixture was shaken gently at room temperature for 90 minutes.The tube was centrifuged, and the original supernatant and fivesuccessive washes (1 mL of pH 7.5 buffer) were collected and their ¹²⁵ Icontent determined using a Packard Auto-Gamma Scintillation SpectrometerModel 5230. The original supernatant exhibited 42,415 cpm (abovebackground); first wash: 6722; second wash: 836; third wash: 202; fourthwash: 48; and fifth wash: 18 cpm. Ethanolamine (400 microliters of 0.5Min pH 7.5 phosphate buffer) was added and shaken with the beads for 90minutes to react with all the remaining azlactone residues. Finally,after an additional four washes with buffer solution the beads and thereaction vessel were counted and exhibited 7002 and 1865 cpm,respectively. This correlates to a level of 2.54 micrograms of ProteinA/10 mg of beads.

A CONTROL experiment was conducted in the same manner except the orderof addition of Protein A and ethanolamine was reversed. The CONTROLexhibited a level of 0.14 microgram of Protein A/10 mg of beads.

In a similar fashion, the effects of the catalyst DBU were examined,with the DBU (25 microliters) being added to the initial Protein Abuffer solution. The result was a level of 3.90 micrograms of ProteinA/10 mg of beads.

EXAMPLE 15

This example illustrates that the Protein A is not just adsorbed oradhering to the beads in some fashion but is actually covalently boundto the polymer beads. The amount of covalently bound protein may beestimated by determining the amount of protein resistant to sodiumdodecylsulfate (SDS) treatment. SDS denatures protein so that only thosemolecules which are covalently bound will remain attached to the beads.

In this experiment, the polymer beads (10 mg) of EXAMPLE 14 (having 3.90micrograms Protein A/10 mg of beads) were incubated with 1% sodiumdodecylsulfate (SDS) (500 microliters) at 37° C. for two hours, followedby centrifugation, and five buffer (550 microliters; pH 7.5) washes.Analysis of the radioactivity of the beads revealed that 73% of theprotein remained attached to the beads.

EXAMPLE 16

This example illustrates that the Protein A attached to the polymerbeads remains active and is not denatured in the attaching process.

Biologically active Protein A can be assayed by determining the amountof antibody which it can bind. Antibody (IgG) conjugated with an enzymemarker, alkaline phosphatase was purchased from Cooper Biomedical(Malvern, Pa.).

In this experiment, 1.0 mg of the polymer beads of EXAMPLE 5 werereacted with unlabeled Protein A and ethanolamine as described inEXAMPLE 14. The beads were reacted with the enzyme-antibody conjugatefor 2 hours. After centrifugation and washing steps, Protein A andCONTROL beads were resuspended in the alkaline phosphatase assaysolution (0.1M sodium glycinate, 1.0 mM ZnCl₂, 1.0 mM CaCl₂, 6.0 mMp-nitrophenyl phosphate, pH 10.4) and rocked continuously to promotemixing. Every 10 min the absorbance of the supernatant solution wasdetermined at 405 nm. The absorbance of the TEST beads increasedlinearly at 5 to 15 times the CONTROL rate, depending on the amount ofimmobilized Protein A. This showed that the protein remained active.

EXAMPLE 17

This example teaches that the attaching reaction with a protein inaqueous media is rapid.

The beads of Example 6 were reacted with radiolabeled Protein A asdescribed in Example 14 except that the quenching and washing steps wereinitiated at various times from 5-180 min. The "zero time" was preparedby addition of the quencher ethanolamine first. The reaction wasperformed in pH 8.5, 20 mM sodium pyrophosphate buffer with DBU. It wasobserved that at 5 minutes 1.34 micrograms of Protein A/10 mg of beadswere bound. This was 80% by weight of the amount bound at 180 minutes.

EXAMPLE 18

This example teaches that a substantially greater concentration ofmultifunctional monomers is required to achieve a low degree of swellingwith hydrophilic polymer beads than with typical hydrophobic macroporouspolymer beads which are essentially non-swelling with difunctionalmonomer concentrations of greater than 20 weight percent.

Employing the procedure of Example 1 with the modification of using 60mL of DMF cosolvent in step 1, two bead formulations were prepared:DMA:PIP:VDM (42:16:42) [52.5:10.1:37.4] and MBA:PIP:VDM (42:16:42)[41.6:12.5:46.0], in which PIP represents N,N'-bis(acryloyl)piperazineprepared by the method of M. C. Tanzi, et al., Biomaterials, 5, 357(1984). In the first set of beads the difunctional monomer molarconcentration was 10.1% and in the second set 54%. In graduatedcylinders, 0.5 mL (dry volume) of the beads of each set were coveredwith the pH 7.5 buffer solution. Within 20 minutes, the beads containing10.1% difunctional monomer had swelled to 3 mL or six times its dryvolume, whereas the 54% crosslinked beads had swelled to 1 mL or onlytwice its dry volume.

Because of their low degree of swelling these beads are especiallyuseful for the preparation of chromatographic supports.

EXAMPLE 19

This example teaches that exceptionally high binding capacities can beachieved with highly crosslinked, azlactone-functional beads and,further, that a surprisingly non-linear relationship exists betweencapacity and azlactone content (i.e., in certain ranges, a modestincrease in reactive group functionality results in an enormous increasein binding capacity).

Two bead formulations were prepared as in Example 18 consisting ofMBA:PIP:VDM:DMA (42:16:10:32) [36.4:10.9:9.6:43.1] and MBA:PIP:VDM:DMA(42:16:30:12) [39.4:11.8:31.2:17.5]. These preparations along with theMBA:PIP:VDM (42:16:42) [41.6:12.5:46.0] beads of Example 18 containrelatively high levels, i.e., 47-54%, of difunctional monomers. Thethree azlactone-functional beads were challenged with radiolabeledProtein A (125 mg/g of beads). Similarly treated was a commercial,oxirane-functional bead preparation, Eupergit-C™ (available from RohmPharma, Weiterstadt, West Germany). (Eupergit-C is believed to be awater swellable, crosslinked polymer bead protected by U.S. Pat. No.4,070,348.) After washing as in Example 14, the levels of bound ProteinA were observed with the various bead preparations as shown in TABLEIII, below.

                  TABLE III                                                       ______________________________________                                                       [Reactive Group]                                                                           Bound Protein A*                                  Bead Sample    (meq/g)      (mg/g)                                            ______________________________________                                        MBA:PIP:VDM:DMA                                                                              0.72         3.6                                               (42:16:10:32)                                                                 [36.4:10.9:9.6:43.1]                                                          MBA:PIP:VDM:DMA                                                                              2.15         11.3                                              (42:16:30:12)                                                                 [39.4:11.8:31.2:17.5]                                                         MBA:PIP:VDM    3.02         54.4                                              (42:16:42)                                                                    [41.6:12.5:46.0]                                                              EUPERGIT-C     0.8-1.0**    9.6                                               ______________________________________                                         *All bound Protein A amounts were greater than 95% SDS resistant.             **According to information provided by the vendor.                       

It was surprising to note that an increase of 40% in reactive groupfunctionality from the 30 wt % VDM to the 42 wt % VDM was accompanied byan enormous 380% increase in weight of bound protein. The waterswellable Eupergit-C product even when projected at equivalent reactivegroup concentration would bind only from 30-36 mg of Protein A per gramof polymer bead.

EXAMPLE 20

This example teaches that the highly crosslinked, azlactone-functionalbeads can bind considerably more protein than oxirane beads.

MBA:PIP:VDM (42:16:42) [41.6:12.5:46.0] beads of Example 18 andEupergit-C™ beads were reacted with 20-500 mg of radiolabeled Protein Aper gram of bead. After washing as described in Example 14, the amountsof bound protein were determined. These results were plotted by a methodoriginally described by Klotz (I. M. Klotz, in "The Proteins", eds. H.Neurath and K. Bailey, Academic Press, Vol. 2, p. 727, 1958) in whichthe inverse of the Protein A bound is plotted versus the inverse of theProtein A concentration. This method is useful for determining themaximum capacity of a binding group for a ligand by extrapolation toinfinite ligand concentration. The maximum binding capacity ofEupergit-C™ for Protein A was 13.5 mg/gram of bead, much lower than the245 mg/gram maximum binding capacity of VDM-containing bead.Additionally, SDS treatment as described in Example 15 reveals that 87%of the Eupergit-C™ Protein A was covalently attached compared with 96%of the VDM Protein A.

EXAMPLE 21

This example teaches that azlactone-functional beads can perform well asa stationary phase in high performance (pressure) liquid chromatography(HPLC).

Eupergit-C™ and MBA:PIP:VDM (42:16:42) [41.6:12.5:46.0] of Example 18were each packed into identical 3×50 mm glass HPLC columns and subjectedto a regimen of increasing flow rates using a Pharmacia FPLC liquidchromatography pumping system. At a flow rate of 1 mL/min the backpressures observed were 1.0 megapascal (MPa) (Eupergit-C™) and 0.8 MPa(azlactone), and at 2.5 mL/min the pressures were 1.6 and 1.3 MPa,respectively. Neither column bed appeared compressed during the lengthytesting period.

EXAMPLE 22

This example teaches that a column of Protein A immobilized ontoazlactone-functional beads can function as an affinity column forImmunoglobulin G (IgG) in a high flow system such as might be useful intreatment of a cancer patient.

Protein A was immobilized onto the MBA:PIP:VDM (42:16:42)[41.6:12.5:46.0] beads of Example 18 as described in Example 13, and a3×37 mm HPLC column was prepared and equilibrated at pH 7.5 in 25 mMsodium phosphate buffer. Human blood serum (0.5 mL), diluted 10-foldwith buffer, was injected into the column at 0.5 mL/min (2 columnvolumes/min). After 8 column volumes the column was washed with 1.0MNaCl in the phosphate buffer to remove any non-specifically boundprotein. Finally, the column was eluted with 1.0M sodium glycinatebuffer, pH 3.0, to remove the bound IgG. 200 μg of IgG eluted from thecolumn which yields a useful capacity of 0.6 moles of IgG bound per moleof Protein A immobilized to azlactone-functional beads.

EXAMPLE 23

This example teaches the preparation of a highly crosslinked beadpolymer of the invention by the reverse phase suspension polymerizationmethod using monomeric 2-vinyl-4,4-dimethylazlactone instead of itsprecursor (PROCESS II).

Preparation ofCopoly(Methylenebisacrylamide:2-Vinyl-4,4-dimethylazlactone) (58:42)[55.5:44.5]

A 3-liter creased, round bottomed flask equipped with a mechanicalstirrer (stirring rate 300 rpm), nitrogen inlet, thermometer, andcondenser was charged with heptane (1043 mL) and carbon tetrachloride(565 mL). This solution was stirred and sparged with nitrogen for 15minutes. A separate solution was prepared as follows:Methylenebisacrylamide (29 grams, 0.188 mole) was dissolved indimethylformamide (160 mL); after solution was achieved, water (160 mL)was added and the resulting solution sparged with nitrogen for 15minutes. Sorbitan sesquioleate (3 mL) was added and sparging continuedfor an additional 5 minutes. At this point, ammonium persulfate (1 gram)was added and sparging continued for 1 minute more. The solution wasthen quickly added to the organic suspending medium. Immediatelyfollowing this addition, VDM (21 grams, 0.151 mole) was added and the,whole mixture was sparged for an additional 5 minutes.N,N,N',N'-tetramethyl-1,2-diaminoethane (2 mL) was added and thereaction temperature rose fairly rapidly from 22 to 29 degrees C. Thereaction mixture was stirred for a total of 4 hours from the time of thediamine addition, then filtered using a "D" (greater than 21micrometers) sintered glass funnel. The filter cake was washed on thefilter with acetone (2×500 mL), then dried at 60 degrees C. and lessthan 1 torr for 24 hours. IR analysis indicated a strong azlactonecarbonyl absorption.

EXAMPLE 24A

A reverse phase suspension polymerization (PROCESS II) was conducted bya procedure similar to that of Example 23 except that a polymericstabilizer was employed instead of the sorbitan sesquioleate. Thepolymeric stabilizer was prepared as follows: A solution ofcopoly(isooctylacrylate:VDM) (95:5) (31.68 grams of a 38.7% solidssolution in ethyl acetate) was diluted with acetone (20.54 grams). Tothis solution was added choline salicylate (1.06 grams, 0.0044 mole) and1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) (0.1 gram) and the resultantmixture was heated at 55 degrees C. for 15 hours. IR anaylsis indicatedthe formation of the desired reaction product.

In the preparation of the bead polymer, 6.0 grams of the above polymericstabilizer solution was utilized. After conducting the polymerization asdescribed in Example 23, isolating and drying the bead polymer, IRanalysis indicated that azlactone groups had all been hydrolyzed.Cyclization was accomplished using acetic anhydride as in Step 2 ofExample 1. The beads were then filtered, washed with ethyl acetate(2×500 mL) and diethyl ether (1×500 mL), then dried under pump vacuum(<1 torr) for 24 hours at 65 degrees C.

EXAMPLE 24B

A reverse phase suspension polymerization was conducted by a proceduresimilar to that of Example 24A except that the polymeric stabilizer usedwas copoly(isooctylacrylate:N-acryloylmethylalanine, sodium salt)(90.5:9.5). IR analysis of the beads obtained indicated that nohydrolysis of the azlactone ring had occurred during the polymerization.

EXAMPLES 24C, 24D, AND 24E

Reverse phase suspension polymerizations were conducted by the procedureof Example 24A except that the polymeric stabilizer used was (24C) 90:10copoly(isooctylacrylate: acrylic acid, sodium salt), (24D) (90:10)copoly(isooctylacrylate: acrylic acid), and (24E) (91.8:8.2)copoly(isooctylacrylate: N-acryloyl- α-aminoisobutyramide).

In all cases, IR analysis of the beads obtained indicated that nohydrolysis of the azlactone ring had occurred during polymerization.

EXAMPLE 25A

The following examples teach preparation of azlactone-functional beadpolymers by dispersion polymerization in alcoholic media (PROCESS III).

Preparation of Copoly(2-Vinyl-4,4-dimethylazlactone: EthyleneglycolDimethylacrylate) (42:58)

A 1-liter creased, round bottomed flask equipped as in Example 23 wascharged with t-butyl alcohol (400 mL), polyvinylpyrrolidone (7.0 grams)and Aliquat™ 336 (2.0 grams) (avaiable from Henkel Corp., Kankakee,Ill.). VDM (21 grams), ethyleneglycol dimethacrylate (EGDMA, 29 grams),and azobis(isobutyronitrile) (AIBN) (0.5 gram) were mixed, and theresultant solution added to the reaction vessel. Nitrogen gas wasbubbled through the stirred reaction mixture for ten minutes, then theflask was immersed in an oil bath preequilibrated to 70 degrees C.Stirring and heating was continued for 2.5 hours, then the mixture wasallowed to cool and was filtered. The filter cake was washed two timeswith acetone and dried at 50 degrees C. in a circulating air oven togive 43 grams of colorless polymer beads. IR analysis showed theazlactone ring to be intact, and that no apparent reaction with thealcohol solvent had taken place.

Additional examples of polymer beads were prepared by procedures similarto those described in this example except that different dispersingsolvents, different monomer ratios, or different comonomers, wereutilized. Beads were separately prepared using isopropanol, ethanol, andmethanol as the dispersing solvent and methylmethacrylate, hydroxyethylmethacrylate, and 2-isopropenyl-4,4-dimethylazlactone were used asmonomers. In all cases, IR analysis indicated that the azlactone ringwas intact in the product beads. Table IV shows the various formulationsprepared by this method.

                  TABLE IV                                                        ______________________________________                                        Polymer Beads Prepared By Dispersion Polymerization                           Monomer Composition (wt %)                                                    Example                                                                              VDM        EGDMA     Other    Solvent                                  ______________________________________                                        25A     48        52        --       t-butanol                                25B    100        --        --       i-propanol                               25C     10        90        --       i-propanol                               25D     20        80        --       i-propanol                               25E     30        70        --       i-propanol                               25F     60        40        --       i-propanol                               25G     80        20        --       i-propanol                               25H     90        10        --       i-propanol                               25I     42        20        38 (MMA)a                                                                              i-propanol                               25J     40        50        10 (HEMA)b                                                                             i-propanol                               25K    100 (IDM)c --        --       i-propanol                               ______________________________________                                         a = methyl methacrylate.                                                      b = 2hydroxyethyl methacrylate                                                c = 2isopropenyl-4,4-dimethylazlactone                                   

EXAMPLE 26

Polymeric beads from the previous preparations were evaluated for theirability to bind Protein A using labeled protein. Radioiodinated ProteinA (Example 14) was diluted with unlabeled Protein A to give a targetspecific radioactivity of 2000 cpm/μg of protein dissolved in phosphatebuffered saline (PBS) 25 mM sodium phosphate, 150 mM NaCl buffer, pH7.5, with a final protein concentration of 250 μg/mL. In triplicatesamples 200 μL of Protein A solution was added to 10 mg of beads andallowed to react at room temperature for one hour. The reaction wasterminated by removal of the protein solution and addition of 500 μL of3.0M ethanolamine, pH 9.0, for quenching the unreacted azlactone sites.After 30 minutes, the beads were washed three times with 500 μL of PBS,transferred to clean test tubes, and monitored for bound radioactiveprotein as in EXAMPLE 14. The following day, the beads were incubatedwith 500 μL of 1% SDS (sodium dodecylsulfate) in PBS for 4 hours at 37degrees C. to remove non-covalently bound protein. After incubation, thebeads were rinsed three times with 500 μL SDS, and the remainingradioactivity was determined. The data from those experiments is shownin TABLE V, below.

                  TABLE V                                                         ______________________________________                                        Properties of Azlactone Polymeric Beads                                                     Protein A SDS                                                   Bead          binding   resistance                                            (Example #)   (mg/g)    (%)                                                   ______________________________________                                        23            3.2       81                                                    24A           2.8       90                                                    25A           1.1       96                                                    25E           4.1       96                                                    ______________________________________                                    

The data of TABLE V show that beads prepared by PROCESSES II and IIIexhibited high protein binding capacities similar to those obtained inPROCESS I.

EXAMPLE 27

The surface area and pore size of azlactone polymer beads prepared bythe methods of Examples 18, 23, and 25A were measured by standardmethods, BET-method (isothermal nitrogen absorption) and mercuryporosimetry, respectively. Reactive groups were determined by modifyingthe method of Example 3 such that the beads were reacted in 0.1M NaOHovernight before titration with standard acid solution. The results,shown in Table VI, indicate that beads prepared by the methods ofExamples 18 and 23 produce materials which were significantly moreporous than Eupergit-C (values from vendor's information) while thebeads produced by the method of Example 25A were nearly nonporous.

                  TABLE VI                                                        ______________________________________                                        Properties of Polymeric Beads                                                        Surface                Reactive                                                                             Reactive                                        area      Ave. Pore    groups density                                  Bead   (m.sup.2 /g)                                                                            size (A)     (meq/g)                                                                              (μeq/m.sup.2)                         ______________________________________                                        Ex. 18 275       650            3.02   11                                     Ex. 23 200       550            2.79   14                                     Ex. 25A                                                                              2.85      25000   (2.5 μm)                                                                          0.19   67                                     Euper- 183       350            0.8-1.0                                                                              5.5                                    git-C                                                                         ______________________________________                                    

The measured average pore size of the porous azlactone beads of Example18 and 23 were somewhat larger than Eupergit-C. The large measured poresize (2.5 micrometers) of the azlactone beads of Example 25A is believedto represent the interstitial volume between beads and indicates a lackof a porous structure. The number of reactive groups available on porousazlactone beads was higher than that available on Eupergit-C which couldbe of great utility when used for chromatographic purposes. Whencompared as reactive groups per unit area, the nonporous azlactone beadsand porous beads were clearly superior to Eupergit-C.

EXAMPLE 28

This example demonstrates the preparation of azlactone-functionalpolymers which have enhanced coating properties for organic andinorganic substrates (PROCESS IV).

A homopolymer of VDM was prepared by free radical polymerization of themonomer using 2,2'-azobisisobutyronitrile (AIBN) as initiator at 30%solids in methyl ethyl ketone (MEK). Samples of this polymer solutionwere reacted with methanol using DBU (2-5 mole % based on methanolcharged) as catalyst. The amount of methanol used was chosen so as toleave a certain fraction of the azlactone rings of the polymer intact.The homopolymer and five modified polymers with 100, 80, 60, 40, 20, and0 molar % remaining were prepared. These will be referred to as VDM-100,VDM-80, VDM-60, VDM-40, VDM-20, and VDM-0, respectively, in succeedingexamples.

Similarly ethylamine was reacted with a fraction of the homopolymersolution to produce a 60% ethylamide/40% azlactone polymer (VDM-60N).

EXAMPLE 29

This example shows the superior coating properties of modified poly VDM(PROCESS IV).

Glass microscope slides (2.5×7.6 cm); SurgiPath™) were dipped threetimes in a 10% solution of VDM-60 or VDM-100 (Example 28) in ethylacetate and allowed to air dry for one hour at room temperature (PROCESSIV). The slides were then immersed in distilled water for 5 to 15minutes and observed for remaining VDM coating. After rinsing, slidescoated with VDM-60 polymer showed evidence of the undisturbed VDMpolymer coating by the appearance of a faint opalescent sheen. A polymerfilm was observed to float off the surface of those slides coated withVDM-100. This indicates that the partially methanol reacted azlactonepolymer binds substantially better to a glass surface than the unreactedpolymer. Similarly glass slides were dip coated in 10% solutions ofVDM-100 and VDM-60N and air dried. Pairs of slides were immersed for 10and 40 minutes in 0.1M HCl solution. The VDM-100 coating bubbled andlifted in each case while the VDM-60N coating remained attached andunchanged by the treatment.

To confirm these qualitative observations, sections of plain glassmicrohematocrit capillary tubing (American Scientific Products, McGawPark, Ill.) were coated with VDM-60 (Example 28) and allowed to air dry(1.2mm internal diameter, 6 mm length; 2 sections per assay 0.90 cm²total surface area). These samples (in triplicate) were incubated withradioiodinated Protein A as previously described (Example 26). Theresults (see Table VII, below) show that azlactone-functional polymercoating of the glass increased both the amount of covalently boundprotein (17-fold greater than non-treated glass) and the % SDSresistance (4.5-fold increase). In addition, incubation in the aqueousPBS solution at room temperature for 16 hours resulted in no significantloss of bound protein, indicating minimal dissolution of theazlactone-functional polymer coating by aqueous solution over thisextended time interval.

                  TABLE VII                                                       ______________________________________                                        The Binding of Protein A to VDM-60 Coated Glass                                             Covalently SDS                                                                bound protein                                                                            resistance                                           Treatment     (ng/cm.sup.2)                                                                            (%)                                                  ______________________________________                                        none          11         16                                                   solvent       26         23                                                   VDM-60        194        72                                                   ______________________________________                                    

EXAMPLE 30

This example illustrates the coating of silica gel withazlactone-functional polymer and the ability of the coated product tocovalently bind protein (PROCESS IV).

Chromatographic grade silica beads (Silicar CC-4™, Mallinkrodt Chemical,St. Louis, Mo.) were dried in an oven at 135 degrees C., <1 torr, for 24hours. A sample of this silica gel (9.81 g) was mixed with a 30% solidssolution of VDM-100 polymer from Example 28 in MEK (1.63 g). Thismixture was covered with more MEK (50 mL) and allowed to stand at roomtemperature for 3 hours with occasional swirling. The solvent was thenremoved under reduced pressure and the coated silica gel, now containing5% by weight azlactone polymer, was dried at <1 torr at room temperatureovernight. Similarly VDM-80, VDM-60, VDM-40, VDM-20, and VDM-0 werecoated onto silica gel. Approximately 10 mg of each coated silica geland untreated silica gel were exposed to labeled Protein A and evaluatedfor protein binding as in Example 26.

The results of radioactivity monitoring of the protein-treated beads andsubsequent SDS treatment are shown below in TABLE VIII.

                  TABLE VIII                                                      ______________________________________                                        The Binding of Protein A to                                                   Azlactone-Functional Polymer-Coated Silica Beads                                             Bound     SDS                                                                 Protein A Resistance                                           Bead Type      (mg/g)    (%)                                                  ______________________________________                                        Untreated      0.15      42                                                   Silica                                                                        VDM-0          0.14      41                                                   VDM-20         1.97      94                                                   VDM-40         2.44      97                                                   VDM-60         2.60      98                                                   VDM-80         2.64      98                                                   VDM-100        2.50      97                                                   ______________________________________                                    

The data of TABLE VIII show coating the beads with polymer containing20% residual azlactone (VDM-20) resulted in a 14-fold increase in theamount of protein bound, with SDS resistance more than doubling.Solutions of polymer containing 40% azlactone functionality or higheryielded protein binding at a 17-fold increase over the uncoated silica.The SDS resistance increased to 98% as a result of theazlactone-functional polymer coating on this inorganic support material.

EXAMPLE 31

This example illustrates the ability of partially reaction azlactonepolymer to coat zirconium oxide ceramic beads (PROCESS IV).

Zirconium oxide ceramic beads (12.65 g) prepared as described in M. P.Righey, Ph.D. Thesis, "The Development of Porous Zirconia as a Supportfor Reverse Phase High Performance Liquid Chromatography", University ofMinnesota, Minneapolis, Minn. (1988) were coated as in Example 30 withVDM-60 to a final 5 weight % loading. Coated and uncoated beads wereevaluated for protein binding using radioiodinated Protein A.

Approximately 20 mg of each bead type (in triplicate) were incubatedwith 400 μL of a dilute solution of labeled Protein A (specificradioactivity at 2000 cpm/μg; 250 μg protein/mL) for one hour, at whichtime 800 μL of 3.0M ethanolamine, pH 9.0, was added to the reactionmixture. After centrifugation and removal of the supernatant, anadditional 800 μL of quenching amine was incubated with the beads for 30minutes. This quench was followed by 3×800 μL rinses with PBS. The beadswere then monitored for radioactivity (10 minutes per tube), and weresubjected to 1% SDS treatment on the following day (see Example 26).

Azlactone-functional polymer coating of the beads resulted in a 24-foldincrease in protein bound to the ceramic material, 0.72 μg protein permg beads versus 0.03 μg per mg for uncoated beads . In addition, thebound protein was greater than 99% resistant to SDS treatment,indicating covalent bond linkage of the protein to theazlactone-functional polymer-coated zirconium oxide support.

EXAMPLE 32

This example illustrates the preparation of a nylon membrane coated withpolyazlactone and the binding of protein to it (PROCESS IV).

Small disks of nylon filtration membrane (Filter-Pure™ Nylon-66, 0.2 μm;Pierce, Rockford, Ill.) were cut (1/4" diameter; total surface area of0.61 cm² /disk) and immersed in a 1% solution of VDM-60 (Example 28) inethyl acetate for one hour at room temperature, followed by air drying.Protein A binding was evaluated by the procedure of Example 26 withexception that polyazlactone-coated disk and untreated disks (intriplicate) were treated with 250 μL of labeled Protein A solution andallowed to react at room temperature for one hour, with the initial30-minute incubation performed under vacuum to ensure complete wettingof the membrane. The reaction was terminated by removal of the proteinsolution and addition of 500 μL of 3.0M ethanolamine, pH 9.0, forquenching the unreacted azlactone sites as before.

The VDM-60 treated nylon membrane disks showed a 60% increase incovalently bound protein over the untreated disks (757 ng Protein A/cm²vs. 472 ng/cm²), with a significant six-fold increase in the % SDSresistance (71% compared to 11%). These results indicate that proteinbinding to this membrane could be increased by a simple treatment withthe azlactone-functional polymer.

EXAMPLE 33

This example demonstrates the coating of polyethylene particles withpartially reacted azlactone polymer and the ability of the coatedorganic particle to bind protein (PROCESS IV).

High density porous polyethylene particles (prepared as disclosed inU.S. Pat. No. 4,539,256) were cryogenically ground according to theprocedure disclosed in Australian Patent No. 551,446 and then coatedwith a 5% loading of VDM-60 by a procedure similar to that of Example30. Untreated and VDM-60 treated particles were evaluated for Protein Abinding as previously outlined (Example 26), using 10 mg of particlesper tube, 200 μL of Protein A solution, and 500 μL of quenching reagent,PBS, and SDS solution.

Radioactive determinations indicated a 65% increase in covalently boundprotein on the VDM-60 treated particles compared to the untreatedpolyethylene. SDS resistance increased from 24% to 65%, representing atwo- to three-fold increase in the relative amounts of covalent proteinbinding.

EXAMPLE 34

This example shows that graft polymers of VDM and polystyrene can beused to coat glass beads (Corning Glass Works, Corning, N.Y.) (PROCESSIV).

Controlled pore glass (CPG) beads were treated as in Example 30 withsufficient 1% solution in toluene of a VDM-grafted polystyrene (2% VDM)to result in a 1% loading of the CPG after evaporation. As a control,ungrafted polystrene was also coated onto CPG by a similar procedure.The coated CPGs were then tested for Protein A binding in comparisonwith the untreated glass material, as previously outlined in Example 26,using 10 mg of support material per tube.

Determination of covalently bound radioactive protein indicated anine-fold increase of total protein binding to the polystyrene-coatedbeads over the uncoated beads, with an additional three-fold increase onthe VDM-grafted polystyrene coated material. SDS results showed adoubling of percent resistance of the bound protein on the VDM-graftedpolystyrene compared with the plain polystyrene-coated beads. These dataindicate a significant increase in covalent protein binding as a directresult of the VDM-grafted polymer coated on the glass beads.

EXAMPLE 35 Derivatization of VDM Copolymer Beads and Demonstration ofHydrophobic Interaction Properties

VDM copolymer beads from Example 18 were derivatized for 96 hours atroom temperature with 0.5M phenethylamine (Aldrich Chem. Co., Milwaukee,Wis.) in 25 mM phosphate buffer, pH 7.5. The beads were washedexhaustively with phosphate buffer, and packed manually into a 0.35 mLOmni™ glass column (3 mm×5 cm; Rainin Instrument Co., Woburn, Mass.).The column was equilibrated on the FPLC system (Pharmacia Inc.,Piscataway, N.J.) with 1.7M ammonium sulfate in 50 mM phosphate buffer,pH 6.8. Ovalbumin (5 mg/mL; Sigma Chem. Co., St. Louis, Mo.) dissolvedin the ammonium sulfate buffer was loaded onto the column. At a flowrate of 1.0 mL/min, a 15 mL gradient (1.7M ammonium sulfate to 0.0M inphosphate buffer) was performed to evaluate elution of the protein byhydrophobic interaction. In this example, 0.53 mg of the protein waseluted late in the gradient at 10.45 mL elution volume (1.16M ammoniumsulfate), with the remaining protein recovered in the void volume. Thisresult is in contrast to a control column using the identical solventsystem, but packed with VDM beads that had been hydrolyzed in phosphatebuffer. The control showed no interaction with the ovalbumin, i.e., allof the protein passing unretarded through the hydrolyzed copolymercolumn at the void volume.

Bovine serum albumin (BSA; Sigma) at 25 mg/mL in ammonium sulfate bufferwas injected onto the column and eluted by the same gradient as above,but at a flow rate of 0.3 mL/min. This high concentration (5 mginjection) of protein resulted in 32% of the total protein being elutedat 10.53 mL (1.17M salt), and the rest recovered in the void volume.Upon reinjection, a fraction of the unbound protein from the void volumebound to the column and eluted at 10.49 mL, suggesting that the initialinjection had over-loaded the column. These data suggest that as thesalt concentration is reduced, the proteins elute from the matrix basedon their hydrophobicity. This demonstration indicates the potentialapplication of the VDM copolymer in hydrophobic interactionchromatography.

EXAMPLE 36 Demonstration of Ion Exchange Properties

VDM copolymer beads from Example 18 were derivatized with 0.7M taurine(Aldrich Chem., Milwaukee, Wis.) in 25 mM phosphate buffer, pH 7.5, for72 hours at room temperature. The excess reagent was rinsed from thebeads with buffer before packing in a 0.35 mL Omni™ glass column (3 mm×5cm). The column was equilibrated with 50 mM acetic acid, pH 5.0. BSA wasdissolved in the equilibration buffer, and 0.8 mg was injected onto thecolumn. After loading the protein in 5 mL of equilibration buffer at aflow rate of 0.5 mL/min, a 25 mL salt gradient from 0 to 2M NaCl (in 50mM acetate, pH 5.0) was applied for ion exchange elution. As detected byUV absorbance readings at 280 nm, 0.54 mg of the BSA eluted from thecolumn at an elution volume of 8.8 mL. The remaining protein wasrecovered in the void volume.

The above protocol was performed with bovine IgG (Sigma) as theexperimental protein to evaluate differences in elution between twoproteins. After loading 0.68 mg IgG, approximately 0.52 mg (64%) waseluted at 8.4 mL, with the remaining IgG recovered in the void volume.These results correlate with previous experiments using commercial ionexchange matrices, in which IgG eluted slightly before BSA on a Mono Scolumn (Pharmacia Co., Piscataway, N.J.).

A mixture of the above two proteins was resolved using thetaurine-azlactone column when it was injected onto the column and elutedat 0.2 mL/min according to the following multiple gradient: a 30 mLgradient from 0 to 0.30M NaCl, followed by a 15 mL gradient to 1.0MNaCl, and final cleansing of bound protein with 2M NaCl. The proteinseluted in two main peaks at 17.8 mL (0.13M NaCl) and at 21.5 mL (0.16MNaCl). These elution volumes vary from the previous examples, due to thechange in flow rate.

EXAMPLE 37 Demonstration of Anion Exchange Properties of AzalactoneBeads

VDM polymer beads from Example 18 were reacted with an excess of4-dimethylamino-1-butylamine in acetone for 12 hours at reflux.Following filtration and washing with acetone to remove unreacted amine,the beads were dried under vacuum at 60 degrees C. The beads were thenpacked into a 2.0 mL glass column, 5 mm×10 cm (Pharmacia Fine Chemicals,Uppsalla, Sweden), and equilibrated with 20 mMtris(hydroxymethyl)aminomethane (Tris), pH 8.0. BSA and bovine IgG wereinjected onto the column and eluted with a 20 mL gradient from 0 to 2MNaCl, 20 mM Tris, pH 8.0. The retention volumes for the proteins were17.0 and 21.7, respectively, with a void volume of 4.1 mL. Thisdemonstrates the use of cationic derivative of the VDM polymer beads foranion exchange separations.

EXAMPLE 38 Demonstration of Size Exclusion Properties

VDM polymer beads from Example 18 were hydrolyzed in 10 mM phosphatebuffer at pH 7.5 for >72 hours at room temperature. The beads werepacked in a 2 mL glass column, 5 mm×10 cm, and equilibrated in 50 mMsodium sulfate in phosphate buffer (10 mM), pH 7.2. The materials shownin TABLE IX, below, were dissolved in water and filtered (0.2 μm) priorto injection onto the column:

                  TABLE IX                                                        ______________________________________                                        Separation of Biological Molecules by Size                                    Exclusion Using Porous Polymer Beads                                          Material        MW (Daltons) Elution Volume                                   ______________________________________                                        blue dextran ™                                                                             2,000,000   1.45     mL                                       thyroglobulin   670,000     1.53                                              catalase        247,000     1.63                                              bovine serum albumin                                                                           69,000     1.80                                              myoglobin        17,000     1.98                                              vitamin B-12     1,355      2.25                                              6M sodium sulfate                                                                                142      2.60                                              ______________________________________                                    

Each material (100 μL) was loaded onto the column and eluted in therunning buffer at a flow rate of 0.2 mL/min. Detection of the elutionvolume was by UV absorbance at 280 nm. The 6M sodium sulfate provides anabsorbance deflection due to a refractive index change caused by theincreased salt concentration, and provides a very low molecular weightnon-protein marker.

The graphed results show a linear relationship between the log of themolecular weight and the elution volume. These data are indicative ofwide ranging size exclusion properties of the hydrolyzed VDM polymer.Based on this standard curve, this particular preparation of azlactonecopolymer beads can be used for size exclusion chromatography over amolecular weight range of 4 log units (100 to 1,000,000).

EXAMPLE 39 Size Exclusion Characteristics of Derivatized VDM

VDM copolymer from Example 18 was derivatized with two amine reagents ofshort chain lengths for further size exclusion studies. The beads wereexposed to either 2M ethylamine in phosphate buffer (25 mM, pH 7.5) or0.5M butylamine (in the same buffer system) for at least 72 hours atroom temperature. The excess amines were removed by rinsing in buffer,and individual 5 mm×10 cm columns were prepared. Blue dextran,thyroglobulin, catalase, and bovine serum albumin were loaded onto thecolumns and eluted as described in Example 38. The results againdemonstrate a linear relationship between the log of the molecularweight and the elution volume.

EXAMPLE 40 Demonstration of Reverse Phase Chromatography

Azlactone polymer beads from Example 18 were derivatized by 1Moctylamine in phosphate buffer (25 mM), pH 7.2, for 72 hours at roomtemperature. The beads were washed free of excess alkyl amine withbuffer and were packed in an Omni 3 mm×5 cm glass chromatography column.The 0.35 mL column was equilibrated in 1.7M ammonium sulfate (pH 7.0).Myoglobin was dissolved in the equilibration buffer and 1.25 mg wasloaded onto the column. Elution with a decreasing salt gradient ofammonium sulfate as in Example 35 did not result in any proteinrecovery. The use of typical protein reverse phase elution conditions, a30 mL gradient of 1% trifluoroacetic acid (TFA) in water to 1% TFA in70% methanol, resulted in recovery of the protein. The demonstration wasrepeated with polymer beads reacted with 0.5M methylamine (C1), 2Methylamine (C2) and 0.5M butylamine (C4) with similar results.

Using the beads with C8 groups attached, injections of myoglobin, BSA,and lysozyme were made and eluted with a 30 mL gradient from 1% TFA inwater to 1% TFA in 70% methanol at a flow rate of 0.1 mL/min. Theelution profiles are summarized in Table X.

                  TABLE X                                                         ______________________________________                                        Reverse Phase Separations of Proteins                                         Using Octylamine-Derivatized Copolymer Beads                                              Peak Elution                                                                             Integrated Area                                        Protein     (mL)       (% of Total)                                           ______________________________________                                        Myoglobin   0.61       18      (Void volume)                                              6.1        28                                                                 36.3       50                                                     BSA         0.67       8       (Void volume)                                              26.8       18                                                                 31.8       30                                                                 36.2       43                                                     Lysozyme    0.72       9       (Void volume)                                              33.8       86                                                     ______________________________________                                    

The differences in these elution profiles are indicative of thedifferences in the size and hydrophobic nature of these proteins anddemonstrate the use of VDM beads derivatized with C1 to C8 groups inreverse phase chromatography.

EXAMPLE 41 Reverse Phase Chromatography of Low Molecular WeightMaterials

VDM beads of Example 18 were reacted with excess n-hexadecylamine (C16)in diethyl ether for an hour. The unreacted amine was removed byfiltration and washing with diethyl ether. The derivatized beads werethen dispersed in methanol and packed into a 4.6×100 mm stainless steelHPLC column. After equilibrating in 45/55 methanol/water, injections ofsmall molecule organics were made and eluted at 0.25 mL/min with UVdetection at 280 nm. Table XI summarizes the retention times and furtherdemonstrates the use of alkylamine derivatized VDM polymer beads asreverse phase chromatography supports.

                  TABLE XI                                                        ______________________________________                                        Reverse Phase Separation of                                                   Low Molecular Weight Materials                                                               Retention                                                      Compound       (Vol/void vol)                                                 ______________________________________                                        uracil         1.0                                                            benzophenone   1.57                                                           nitrobenzene   2.15                                                           ______________________________________                                    

EXAMPLE 42 Coating of Azlactone Monomer onto Polystyrene Wells

2-Vinyl-4,4-dimethyl azlactone monomer (VDM) was formulated with 0.25 gby weight of a photoinitiator (IRGACURE™ 651 (Ciba Geigy)), then paintedon the surface of polystyrene microtiter wells (Immulon™ II, Dynatech,Springfield, Va.). The wells were then irradiated under a nitrogenatmosphere using a bank of four fluorescent blacklight tubes (GTESylvania, Inc.) for 30 minutes. This resulted in the polymerization ofthe azlactone monomer on the surface of the wells. Other monomers suchas 4-isopropenyl-4,4-dimethylazlactone (IDM) can be coated in a similarmanner.

EXAMPLE 43 Coating Azlactone-Functional Copolymers Onto PolystyreneWells

Copolymers of VDM and methyl methacrylate (MMA) were prepared bystandard free radical solution polymerization techniques. Samples ofcopolymers containing VDM/MMA ratios (mole/mole) of 85:15, 70:30, and50:50 were each diluted with 10% solids in ethyl acetate. Coating ofmicrotiter wells was accomplished by placing 3 drops of polymer solutioninto each well using a disposable pipette, followed by evaporation in acirculating air oven at 60 degrees C. for 30 minutes. Alternatively,copolymer solution was painted onto the wells, followed by solventevaporation.

EXAMPLE 44 Use of Polystyrene Wells for Improved Immunoassays

Evaluation of the modified surfaces of the wells of EXAMPLE 42 wascarried out by adding aqueous solutions of mouse IgG (Sigma Chemical) oranti-goat IgG (Cooper-BioMedical, Malvern, Pa.) at 50 μg/mL in PBS, pH7.0, to 48 wells and incubating them at room temperature for 2 hours.The solutions were aspirated and a fixing solution of BSA (2.5 mg/mL)and sucrose (5%) in PBS was incubated for 30 minutes. The coated wellswere then aspirated and dried. A similar number of of unmodified wellswere coated by this procedure.

The amount of bound protein was evaluated by adding enzyme-labeledreagents, anti-mouse IgG-alkaline phosphatase and goat IgG-horseradishperoxidase to mouse IgG and anti-goat IgG treated wells, respectively,incubating for 1 hour and washing the wells three times with a pH 7.5buffered non-ionic surfactant solution. After adding the appropriateenzyme substrates, the color produced by the bound enzyme-labeledreagents was measured. The data from these wells are summarized in TABLEXII below.

                  TABLE XII                                                       ______________________________________                                        Binding of Antibodies to Azlactone                                            Copolymer-Coated Microtiter Wells                                             Well Treatment                                                                              Absorbance  SD*    CV** (%)                                     ______________________________________                                        Anti-IgG                                                                      Control (No Treatment)                                                                      0.078       0.029  37                                           Azlactone Treated                                                                           0.095       0.023  25                                           IgG                                                                           Control       0.664       0.082  12                                           Azlactone Treated                                                                           1.161       0.115   9                                           ______________________________________                                         *SD = standard deviation                                                      **CV = coefficient of variation (SD/mean)                                

Wells treated with mouse IgG produced nearly 75% more signal thanuntreated wells while the anti-goat IgG wells produced over a 20%increase in coupled signal. The coefficient of variation of the modifiedwells were about one-third less than those of the untreated wells.

The data of TABLE XII indicates that both the amount of protein boundand the reproducibility of the treatment are increased over the normalpassive adsorption technique.

EXAMPLE 45 Improved Binding of Allergenic Proteins to Coated PolystyreneWells

Perennial ryegrass (PRG) extract and chymopapain (CP) (3M DiagnosticSystems, Santa Clara, Calif.) were isotopically labeled with I-125 andincubated in control (untreated) and VDM/MMA-treated polystyrenemicrotiter wells prepared as in Example 43 for 2 hours at ambienttemperature. Radioactive solution was removed by aspiration andunreacted/unbound sites on the surface were blocked by incubation withserum albumin for 1 hour. The residual radioactivity of the dried wellswas determined and the corresponding amounts of bound protein calculatedas shown in TABLE XIII below.

                  TABLE XIII                                                      ______________________________________                                        Binding of Allergenic Proteins                                                to Coated and Control Polystyrene Wells                                                Bound                                                                Sample   Protein (ng)    SD     CV (%)                                        ______________________________________                                        PRG                                                                           Control  66              2.2    3.4                                           VDM      79              3.8    4.8                                           CP                                                                            Control  45              2.2    4.8                                           VDM      56              2.6    4.7                                           ______________________________________                                    

The data of TABLE XIII show that VDM-coated polystyrene wells bound20-25% more allergen protein with similar precision than the Control.

EXAMPLE 46 Increased Irreversible Binding of Allergenic Proteins toCoated Polystyrene Wells

Microtiter test wells coated with I-125-labeled-allergens, as in EXAMPLE45, were incubated with 0.1% SDS at 37° C. or in phosphate buffer atambient temperature. After 4 hours solutions were removed by aspirationand rinsed twice with deionized water, and the residual bound I-125 wasrinsed twice with deionized water, and the residual bound I-125 wasdetermined. The percent protein remaining is presented below in TABLEXIV.

                  TABLE XII                                                       ______________________________________                                        Increased Resistance of Azlactone-Bound                                       Protein to Solubilization by Denaturant                                                  % Protein Remaining                                                           PRG*        CP**                                                   SAMPLE       BUFFER    SDS     BUFFER  SDS                                    ______________________________________                                        Control      83        43      74      37                                     50/50 VDM/MMA                                                                              85        64      82      68                                     70/30 VDM/MMA                                                                              85        64      85      73                                     85/15 VDM/MMA                                                                              86        67      83      73                                     IDM          83        45      77      49                                     ______________________________________                                         *PRG = perennial rye grass                                                    **CP = chymopapain                                                       

With the possible exception of the isopropenyl derivatives with PRG, thedata of TABLE XIV show there is considerably higher residual binding inazlactone-treated wells than in controls indicating covalent binding ofallergen protein.

COMPARATIVE EXAMPLE 47

The following Example is an attempt to prepare azlactone-functionalbeads by a procedure similar to that of EXAMPLE 3 of U.S. Pat. No.4,070,348.

Heptane (42 g), perchloroethylene (84 g) and benzoyl peroxide (0.5 g)were placed in a 500 mL round bottom flask. Acrylamide (15 g), VDM (15g), ethylene glycol dimethacrylate (0.76 g), and polymeric stabilizer(0.025 g) were dissolved in DMF (20 g) and this solution was then addedto the reaction flask at room temperature. The polymeric stabilizer usedwas an isobutylacrylate/n- butylacrylate/VDM (45:45:10) copolymer whichhad been reacted with choline salicylate by a procedure similar to thatof EXAMPLE 24A. The monomer phase was distributed in the organic phaseby stirring at 350 rpm and was purged with N₂ for 45 minutes. Withexternal cooling, the polymerization was initiated by the addition ofdimethylamiline (0.25 g). Within three minutes, the monomer mixture hadseparated out as a large, crosslinked mass around the stirrer shaft. Nobeads were evident.

EXAMPLE 48 COMPARATIVE

The following is an attempt to prepare an azlactone-functional beadaccording to the teachings of U.S. Pat. No. 4,070,348. The reaction wascarried out using the same ingredients and their amounts as specified inEXAMPLE 25 of U.S. Pat. No. 4,070,348, except that VDM was substitutedfor glycidyl acrylate. Again, a crosslinked polymer mass separatedwithin two minutes of initiation of the reaction. No beads were evident.

Various modifications and alterations of this invention will becomeapparent to those skilled in the art without departing from the scopeand spirit of this invention, and it should be understood that thisinvention is not to be unduly limited to the illustrative embodimentsset forth herein.

We claim:
 1. A crosslinked, azlactone-functional polymer support havingmore than 5 and up to 99 molar parts of multifunctional ethylenicallyunsaturated crosslinking monomer incorporated therein, and wherein saidpolymer support swells less than three-fold in water.
 2. Anazlactone-functional polymer support according to claim 1 having unitsof the formula: ##STR15## wherein R¹ is H or CH₃,R² and R³ independentlycan be an alkyl group having 1 to 14 carbon atoms, a cycloalkyl grouphaving 3 to 14 carbon atoms, an aryl group having 5 to 12 ring atoms, anarenyl group having 6 to 26 carbon and unitary heteroatoms, or R² and R³taken together with the carbon to which they are joined can form acarbocyclic ring containing 4 to 12 ring atoms, and n is an integer 0or
 1. 3. The azlactone-functional polymer support according to claim 2wherein R¹ is hydrogen.
 4. The azlactone-functional polymer supportaccording to claim 2 wherein R² and R³ are methyl.
 5. An adduct polymersupport having units of the formula: ##STR16## wherein R¹, R², R³ and nare as previously defined,n=0, or 1, X is --O--, --S--, --NH--, or##STR17## wherein R⁴ is alkyl or aryl, and G is the residue of HXG whichperforms the complexing, catalyzing, separating, or reagent function ofthe adduct polymer support, said adduct polymer support containinggreater than 5 and up to 99 molar parts of multifunctional ethylenicallyunsaturated crosslinking monomer incorporated therein, and wherein saidpolymer support swells less than three-fold in water.
 6. The adductpolymer support according to claim 5 wherein R¹, R², and R³ are methyland n=0.
 7. The adduct polymer support according to claim 5 wherein HXGis selected from the group consisting of biomacromolecules, catalysts,reagents, and dyes.
 8. An adduct polymer support according to claim 5containing in the range of greater than 20 and up to 99 molar parts ofcrosslinking monomer incorporated therein.
 9. An adduct polymer supporthaving units of the formula ##STR18## wherein R¹, R², R³ and n are aspreviously defined,n=0 or 1, X is --O--, --S--, --NH--, or ##STR19##wherein R⁴ is alkyl or aryl, and G is the residue of HXG which performsthe complexing, catalyzing, separating, or reagent function of theadduct polymer support, said adduct polymer support containing greaterthan 20 and up to 99 molar parts of multifunctional ethylenicallyunsaturated crosslinking monomer incorporated therein and having adegree of swelling in water less than 3 times the unswelled volume. 10.The azlactone-functional polymer support according to claim 1 whereinsaid ethylenically unsaturated crosslinking monomer is present in therange of 7 to 99 molar parts.
 11. The azlactone-functional polymersupport according to claim 1 wherein said ethylenically unsaturatedcrosslinking monomer is present in the range of 10 to 99 molar parts.12. The adduct polymer support according to claim 5 wherein saidethylenically unsaturated crosslinking monomer is present in the rangeof 7 to 99 molar parts.
 13. The adduct polymer support according toclaim 5 wherein said ethylenically-unsaturated crosslinking monomer ispresent in the range of 10 to 99 molar parts.